Controlled release cytotoxic agent compositions and methods for the treatment of otic disorders

ABSTRACT

Disclosed herein are compositions and methods for the treatment of otic diseases or conditions with cytotoxic agent compositions and formulations administered locally to an individual afflicted with an otic disease or condition, through direct application of these compositions and formulations onto or via perfusion into the targeted auris structure(s).

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/076,576 filed Jun. 27, 2008, U.S. Provisional Application No.61/082,450 filed Jul. 21, 2008, U.S. Provisional Application No.61/094,384 filed Sep. 4, 2008, U.S. Provisional Application No.61/101,112 filed Sep. 29, 2008, and U.S. Provisional Application No.61/140,033 filed Dec. 22, 2008, all of which are incorporated herein intheir entirety.

BACKGROUND OF THE INVENTION

Vertebrates have a pair of ears, placed symmetrically on opposite sidesof the head. The ear serves as both the sense organ that detects soundand the organ that maintains balance and body position. The ear isgenerally divided into three portions: the outer ear, auris media (ormiddle ear) and the auris interna (or inner ear).

SUMMARY OF THE INVENTION

Described herein are compositions, formulations, manufacturing methods,therapeutic methods, uses, kits, and delivery devices for the controlledrelease or delivery of at least one cytotoxic agent to at least onestructure or region of the ear. In certain embodiments, the cytotoxicagent is an antimetabolite, an antifolate, an alkylating agent and/or aDNA intercalator. In certain embodiments, the cytotoxic agent is aprotein, an antibody, DNA, a carbohydrate, an inorganic compound, or anorganic compound. In certain embodiments, the cytotoxic agent is anorganic small molecule. In certain specific embodiments, the cytotoxicsmall molecule is methotrexate, cyclophosphamide, or thalidomide, ametabolite, salt, polymorph, prodrug, analogue, or derivative thereof.In certain embodiments, preferred metabolites of cyclophosphamide are4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard, andcombinations thereof.

In some embodiments, the target portion of the ear is the middle ear orauris media. In other embodiments, the target portion of the ear is theinner ear, or auris interna. In other embodiments, the target portion ofthe ear is the middle ear, or auris media. In still other embodiments,the target portion of the ear is both the auris media and the aurisinterna. In some embodiments, the controlled release formulationsfurther comprise a rapid or immediate release component for delivering acytotoxic agent to the auris media and/or the auris interna. Allformulations comprise excipients that are auris-media and/orauris-interna acceptable.

Also disclosed herein are methods for inducing selective cellulartoxicity within the auris media or auris interna comprisingadministration of the cytotoxic agent controlled release formulationsdescribed herein.

Also disclosed herein are methods for the treatment of otic disorderscomprising administration of cytotoxic agent controlled releaseformulations. In some embodiments, the otic disorder is an autoimmuneinner ear disease (AIED). In other embodiments, the otic disorder iscancer of the ear. Cytotoxic agents that are also suitable for thetreatment of cancer of the ear in the formulations and compositionsdisclosed herein include acridine carboxamide, actinomycin,17-N-allylamino-17-demethoxygeldanamycin, amsacrine, aminopterin,anthracycline, antineoplastic, antineoplaston, 5-azacytidine,azathioprine, BL22, bendamustine, biricodar, bleomycin, bortezomib,bryostatin, busulfan, calyculin, camptothecin, capecitabine,carboplatin, chlorambucil, cisplatin, cladribine, clofarabine,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine,dichloroacetic acid, discodermolide, docetaxel, doxorubicin, epirubicin,epothilone, eribulin, estramustine, etoposide, exatecan, exisulind,ferruginol, floxuridine, fludarabine, fluorouracil, fosfestrol,fotemustine, gemcitabine, hydroxyurea, IT-101, idarubicin, ifosfamide,imiquimod, irinotecan, irofulven, ixabepilone, laniquidar, lapatinib,lenalidomide, lomustine, lurtotecan, mafosfamide, masoprocol,mechlorethamine, melphalan, mercaptopurine, mitomycin, mitotane,mitoxantrone, nelarabine, nilotinib, oblimersen, oxaliplatin, PAC-1,paclitaxel, pemetrexed, pentostatin, pipobroman, pixantrone, plicamycin,procarbazine, proteasome inhibitors (e.g., bortezomib), raltitrexed,rebeccamycin, rubitecan, SN-38, salinosporamide A, satraplatin,streptozotocin, swainsonine, tariquidar, taxane, tegafur-uracil,temozolomide, testolactone, thioTEPA, tioguanine, topotecan,trabectedin, tretinoin, triplatin tetranitrate,tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine, vorinostat, and zosuquidar. Alsodisclosed herein is the local delivery of controlled release cytotoxicagent compositions and formulations to suppress or ameliorate auditoryand vestibular impairment as a result of AIED, which may be provoked byother autoimmune conditions, including Ankylosing Spondylitis, SystemicLupus Erythematosis (SLE), Sjögren's Syndrome, Cogan's disease,ulcerative colitis, Wegener's granulomatosis, rheumatoid arthritis,scleroderma and Behçet's disease (also known as Bechet's disease andadamantiades). In other embodiments, the otic disorder is otitis media.

In some embodiments, the compositions described herein are administeredso that the composition is in contact with the crista fenestraecochleae, the round window or the tympanic cavity.

The auris formulations and therapeutic methods described herein havenumerous advantages that overcome the previously-unrecognizedlimitations of formulations and therapeutic methods described in priorart.

Sterility

The environment of the inner ear is an isolated environment. Theendolymph and the perilymph are static fluids and are not in contiguouscontact with the circulatory system. The blood-labyrinth-barrier (BLB),which includes a blood-endolymph barrier and a blood-perilymph barrier,consists of tight junctions between specialized epithelial cells in thelabyrinth spaces (i.e., the vestibular and cochlear spaces). Thepresence of the BLB limits delivery of active agents (e.g., cytotoxicagents) to the isolated microenvironment of the inner ear. Auris haircells are bathed in endolymphatic or perilymphatic fluids and cochlearrecycling of potassium ions is important for hair cell function. Whenthe inner ear is infected, there is an influx of leukocytes and/orimmunoglobins (e.g. in response to a microbial infection) into theendolymph and/or the perilymph and the delicate ionic composition ofinner ear fluids is upset by the influx of leukocytes and/orimmunoglobins. In certain instances, a change in the ionic compositionof inner ear fluids results in hearing loss, loss of balance and/orossification of auditory structures. In certain instances, even traceamounts of pyrogens and/or microbes can trigger infections and relatedphysiological changes in the isolated microenvironment of the inner ear.

Due to the susceptibilty of the inner ear to infections, aurisformulations require a level of sterility (e.g., low bioburden) that hasnot been recognized hitherto in prior art. Provided herein are aurisformulations that are manufactured with low bioburden or sterilized withstringent sterilty requirements and are suitable for administration tothe middle and/or inner ear. In some embodiments, the auris compatiblecompositions described herein are substantially free of pyrogens and/ormicrobes.

Compatibility with Inner Ear Environment

Described herein are otic formulations with an ionic balance that iscompatible with the perilymph and/or the endolymph and does not causeany change in cochlear potential. In specific embodiments,osmolarity/osmolality of the present formulations is adjusted, forexample, by the use of appropriate salt concentrations (e.g.,concentration of sodium salts) or the use of tonicity agents whichrenders the formulations endolymph-compatible and/orperilymph-compatible (i.e. isotonic with the endolymph and/orperilymph). In some instances, the endolymph-compatible and/orperilymph-compatible formulations described herein cause minimaldisturbance to the environment of the inner ear and cause minimumdiscomfort (e.g., vertigo) to a mammal (e.g., a human) uponadministration. Further, the formulations comprise polymers that arebiodegradable and/or dispersable, and/or otherwise non-toxic to theinner ear environment. In some embodiments, the formulations describedherein are free of preservatives and cause minimal disturbance (e.g.,change in pH or osmolarity, irritation) in auditory structures. In someembodiments, the formulations described herein comprise antioxidantsthat are non-irritating and/or non-toxic to otic structures.

Dosing Frequency

The current standard of care for auris formulations requires multipleadministrations of drops or injections (e.g. intratympanic injections)over several days (e.g., up to two weeks), including schedules ofreceiving multiple injections per day. In some embodiments, aurisformulations described herein are controlled release formulations, andare administered at reduced dosing frequency compared to the currentstandard of care. In certain instances, when an auris formulation isadministered via intratympanic injection, a reduced frequency ofadministration alleviates discomfort caused by multiple intratympanicinjections in individuals undergoing treatment for a middle and/or innerear disease, disorder or condition. In certain instances, a reducedfrequency of administration of intratympanic injections reduces the riskof permanent damage (e.g., perforation) to the ear drum. Theformulations described herein provide a constant, sustained, extended,delayed or pulsatile rate of release of an active agent into the innerear environment and thus avoid any variability in drug exposure intreatment of otic disorders.

Therapeutic Index

Auris formulations described herein are administered into the ear canal,or in the vestibule of the ear. Access to, for example, the vestibularand cochlear apparatus will occur through the auris media including theround window membrane, the oval window/stapes footplate, the annularligament and through the otic capsule/temporal bone. Otic administrationof the formulations described herein avoids toxicity associated withsystemic administration (e.g., hepatotoxicity, cardiotoxicity,gastrointestinal side effects, renal toxicity) of the active agents. Insome instances, localized administration in the ear allows an activeagent to reach a target organ (e.g., inner ear) in the absence ofsystemic accumulation of the active agent. In some instances, localadministration to the ear provides a higher therapeutic index for anactive agent that would otherwise have dose-limiting systemic toxicity.

Prevention of Drainage into Eustachian Tube

In some instances, a disadvantage of liquid formulations is theirpropensity to drip into the eustachian tube and cause rapid clearance ofthe formulation from the inner ear. Provided herein, in certainembodiments, are auris formulations comprising polymers that gel at bodytemperature and remain in contact with the target auditory surfaces(e.g., the round window) for extended periods of time. In someembodiments, the formulations further comprise mucoadhesives that allowthe formulations to adhere to otic mucosal surfaces. In some instances,the auris formulations described herein avoid attenuation of therapeuticbenefit due to drainage or leakage of active agents via the eustachiantube.

DESCRIPTION OF CERTAIN EMBODIMENTS

Described herein are controlled release compositions and devices fortreating otic disorders comprising a therapeutically-effective amount ofa cytotoxic agent, a controlled release auris-acceptable excipient andan auris-acceptable vehicle. In one aspect, the controlled releaseauris-acceptable excipient is chosen from an auris-acceptable polymer,an auris-acceptable viscosity enhancing agent, an auris-acceptable gel,an auris-acceptable paint, an auris-acceptable foam, an auris-acceptablemicrosphere or microparticle, an auris-acceptable hydrogel, anauris-acceptable in situ forming spongy material, an auris-acceptableactinic radiation curable gel, an auris-acceptable liposome, anauris-acceptable nanocapsule or nanosphere, an auris-acceptablethermoreversible gel or combinations thereof. In further embodiments,the auris-acceptable viscosity enhancing agent is a cellulose, acellulose ether, alginate, polyvinylpyrrolidone, a gum, a cellulosicpolymer or combinations thereof. In yet another embodiment, theauris-acceptable viscosity enhancing agent is present in an amountsufficient to provide a viscosity of between about 1000 to about1,000,000 centipoise. In still another aspect, the auris-acceptableviscosity enhancing agent is present in an amount sufficient to providea viscosity of between about 50,000 to about 1,000,000 centipoise. Insome embodiments, the cytotoxic agent formulations or compositions areoptimal for osmolality or osmolarity of the target auris structure toensure homeostasis is maintained.

In some embodiments, the compositions are formulated for pH, and apractical osmolality or osmolarity to ensure that homeostasis of thetarget auris structure is maintained. A perilymph-suitableosmolarity/osmolality is a practical/deliverable osmolarity/osmolalitythat maintains the homeostasis of the target auris structure duringadministration of the pharmaceutical formulations described herein. Forexample, the osmolarity of the perilymph is between about 270-300mOsm/L, and the compositions described herein are optionally formulatedto provide a practical osmolarity of about 150 to about 1000 mOsm/L. Incertain embodiments, the formulations described herein provide apractical and/or deliverable osmolarity within about 150 to about 500mOsm/L at the target site of action (e.g., the inner ear and/or theperilymph and/or the endolymph). In certain embodiments, theformulations described herein provide a practical osmolarity withinabout 200 to about 400 mOsm/L at the target site of action (e.g., theinner ear and/or the perilymph and/or the endolymph). In certainembodiments, the formulations described herein provide a practicalosmolarity within about 250 to about 320 mOsm/L at the target site ofaction (e.g., the inner ear and/or the perilymph and/or the endolymph).In certain embodiments, the formulations described herein provide aperilymph-suitable osmolarity within about 150 to about 500 mOsm/L,about 200 to about 400 mOsm/L or about 250 to about 320 mOsm/L at thetarget site of action (e.g., the inner ear and/or the perilymph and/orthe endolymph). In certain embodiments, the formulations describedherein provide a perilymph-suitable osmolality within about 150 to about500 mOsm/kg, about 200 to about 400 mOsm/kg or about 250 to about 320mOsm/kg at the target site of action (e.g., the inner ear and/or theperilymph and/or the endolymph). Similarly, the pH of the perilymph isabout 7.2-7.4, and the pH of the present formulations is formulated(e.g., with the use of buffers) to provide a perilymph-suitable pH ofabout 5.5 to about 9.0, about 6.0 to about 8.0 or about 7.0 to about7.6. In certain embodiments, the pH of the formulations is within about6.0 to about 7.6. In certain instances, the pH of the endolymph is about7.2-7.9, and the pH of the present formulations is formulated (e.g.,with the use of buffers) to be within about 5.5 to about 9.0, withinabout 6.5 to about 8.0 or within about 7.0 to about 7.6.

In some aspects, the controlled-release auris-acceptable excipient isbiodegradable. In some aspects the controlled release auris-acceptableexcipient is bioeliminated (e.g., degraded and/or eliminated throughurine, feces or other routes of elimination). In another aspect, thecontrolled release composition further comprises an auris-acceptablemucoadhesive, an auris-acceptable penetration enhancer or anauris-acceptable bioadhesive.

In one aspect, the controlled release cytotoxic agent composition isdelivered using a drug delivery device, which is a needle and syringe, apump, a microinjection device or combinations thereof. In someembodiments, the cytotoxic agent of the controlled release compositionhas limited or no systemic release, is toxic when administeredsystemically, has poor pK characteristics or combinations thereof. Insome aspects, the cytotoxic agent is a small molecule agent. In otheraspects, the cytotoxic agent is an antibody.

Also disclosed herein is a method for treating an otic disordercomprising administering at least once every 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 days, at least once a week, once every two weeks,once every three weeks, once every four weeks, once every five weeks, oronce every six weeks; or once a month, once every two months, once everythree months, once every four months, once every five months, once everysix months, once every seven months, once every eight months, once everynine months, once every ten months, once every eleven months, or onceevery twelve months with the compositions and formulations disclosedherein. In particular embodiments, the controlled release formulationsdescribed herein provide a sustained dose of cytotoxic agent to theinner ear between subsequent doses of the controlled releaseformulation. That is, taking one example only, if new doses of thecytotoxic agent controlled release formulation are adminstered viaintratympanic injection to the round window membrane every 10 days, thenthe controlled release formulation provides an effective dose ofcytotoxic agent to the inner ear (e.g., across the round windowmembrane) during that 10-day period.

In one aspect, the composition is administered so that the compositionis in contact with the crista fenestrae cochleae, the round windowmembrane or the tympanic cavity. In one aspect the composition isadministered by intratympanic injection.

Provided herein is a pharmaceutical composition or device comprising anamount of a cytotoxic agent that is therapeutically effective fortreating an otic disease or condition, the pharmaceutical composition ordevice comprising substantially low degradation products of thecytotoxic agent, the pharmaceutical composition or device furthercomprising two or more characteristics selected from:

-   -   (i) between about 0.1% to about 10% by weight of the cytotoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate cytotoxic agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation, and    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject.

In some embodiments, a pharmaceutical composition or device describedherein comprises:

-   -   (i) between about 0.1% to about 10% by weight of the cytotoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106; and    -   (iii) multiparticulate cytotoxic agent.

In some embodiments, a pharmaceutical composition or device describedherein comprises:

-   -   (i) between about 0.1% to about 10% by weight of the cytotoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate cytotoxic agent; and    -   (iv) a gelation temperature between about 19° C. to about 42° C.

In some embodiments, any pharmaceutical composition or device describedabove comprises substantially low degradation products of the cytotoxicagent.

In some embodiments a pharmaceutical composition or device describedabove provides a practical osmolarity between about 150 and 500 mOsm/L.In some embodiments a pharmaceutical composition or device describedabove provides a practical osmolarity between about 200 and 400 mOsm/L.In some embodiments a pharmaceutical composition or device describedabove provides a practical osmolarity between about 250 and 320 mOsm/L.

In some embodiments, the cytotoxic agent is released from thepharmaceutical composition or device described above for a period of atleast 3 days. In some embodiments, the cytotoxic agent is released fromthe pharmaceutical composition or device described above for a period ofat least 5 days. In some embodiments, the cytotoxic agent is releasedfrom the pharmaceutical composition or device described above for aperiod of at least 10 days. In some embodiments, the cytotoxic agent isreleased from the pharmaceutical composition or device described abovefor a period of at least 14 days. In some embodiments, the cytotoxicagent is released from the pharmaceutical composition or devicedescribed above for a period of at least one month.

In some embodiments, a pharmaceutical composition or device describedabove comprises cytotoxic agent as a neutral compound, a free acid, afree base, a salt or a prodrug. In some embodiments, a pharmaceuticalcomposition or device described above comprises cytotoxic agent as aneutral compound, a free acid, a free base, a salt or a prodrug, or acombination thereof.

In some embodiments, a pharmaceutical composition or device describedabove is an auris-acceptable thermoreversible gel. In some embodimentsof the pharmaceutical composition or device, thepolyoxyethylene-polyoxypropylene triblock copolymer is bioeliminated.

In some embodiments the pharmaceutical composition or device furthercomprises a penetration enhancer. In some embodiments, thepharmaceutical composition or device further comprises a dye.

In some embodiments, the pharmaceutical composition or device furthercomprises the cytotoxic agent, or pharmaceutically acceptable saltthereof, prodrug or combination thereof as an immediate release agent.

In some embodiments of the pharmaceutical composition or device thecytotoxic agent comprises multiparticulates. In some embodiments of thepharmaceutical composition or device the cytotoxic agent is essentiallyin the form of micronized particles. In some embodiments of thepharmaceutical composition or device, the cytotoxic agent is in the formof micronized cytotoxic agent powder (micro-cytotoxic agent powder).

In some embodiments, a pharmaceutical composition or device describedabove comprises about 10% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.In some embodiments, a pharmaceutical composition or device describedabove comprises about 15% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.In some embodiments, a pharmaceutical composition or device describedabove comprises about 20% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.In some embodiments, a pharmaceutical composition or device describedabove comprises about 25% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.

In some embodiments, a pharmaceutical composition or device describedabove comprises about 0.01% of a cytotoxic agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition. Insome embodiments, a pharmaceutical composition or device described abovecomprises about 0.05% of a cytotoxic agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition. Insome embodiments, a pharmaceutical composition or device described abovecomprises about 0.1% of a cytotoxic agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition. Insome embodiments, a pharmaceutical composition or device described abovecomprises about 1% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition. In someembodiments, a pharmaceutical composition or device described abovecomprises about 2.5% of a cytotoxic agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition. Insome embodiments, a pharmaceutical composition or device described abovecomprises about 5% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition. In someembodiments, a pharmaceutical composition or device described abovecomprises about 10% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition. In someembodiments, a pharmaceutical composition or device described abovecomprises about 20% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition. In someembodiments, a pharmaceutical composition or device described abovecomprises about 30% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition. In someembodiments, a pharmaceutical composition or device described abovecomprises about 40% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition. In someembodiments, a pharmaceutical composition or device described abovecomprises about 50% of a cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, by weight of the composition.

In some embodiments, a pharmaceutical composition or device describedabove has a pH between about 5.5 to about 8.0. In some embodiments, apharmaceutical composition or device described above has a pH betweenabout 6.0 to about 8.0. In some embodiments, a pharmaceuticalcomposition or device described above has a pH between about 6.0 toabout 7.6. In some embodiments, a pharmaceutical composition or devicedescribed above has a pH between about 7.0 to about 7.6.

In some embodiments, a pharmaceutical composition or device describedabove contains less than 100 colony forming units (cfu) ofmicrobiological agents per gram of formulation. In some embodiments, apharmaceutical composition or device described above contains less than50 colony forming units (cfu) of microbiological agents per gram offormulation. In some embodiments, a pharmaceutical composition or devicedescribed above contains less than 10 colony forming units (cfu) ofmicrobiological agents per gram of formulation.

In some embodiments, a pharmaceutical composition or device describedabove contains less than 5 endotoxin units (EU) per kg of body weight ofa subject. In some embodiments, a pharmaceutical composition or devicedescribed above contains less than 4 endotoxin units (EU) per kg of bodyweight of a subject.

In some embodiments a pharmaceutical composition or device describedabove provides a gelation temperature between about between about 19° C.to about 42° C. In some embodiments a pharmaceutical composition ordevice described above provides a gelation temperature between aboutbetween about 19° C. to about 37° C. In some embodiments apharmaceutical composition or device described above provides a gelationtemperature between about between about 19° C. to about 30° C.

In some embodiments, the pharmaceutical composition or device is anauris-acceptable thermoreversible gel. In some embodiments, thepolyoxyethylene-polyoxypropylene triblock copolymer is biodegradableand/or bioeliminated (e.g., the copolymer is eliminated from the body bya biodegradation process, e.g., elimination in the urine, the feces orthe like). In some embodiments, a pharmaceutical composition or devicedescribed herein further comprises a mucoadhesive. In some embodiments,a pharmaceutical composition or device described herein furthercomprises a penetration enhancer. In some embodiments, a pharmaceuticalcomposition or device described herein further comprises a thickeningagent. In some embodiments, a pharmaceutical composition or devicedescribed herein further comprises a dye.

In some embodiments, a pharmaceutical composition or device describedherein further comprises a drug delivery device selected from a needleand syringe, a pump, a microinjection device, a wick, an in situ formingspongy material or combinations thereof.

In some embodiments, a pharmaceutical composition or device describedherein is a pharmaceutical composition or device wherein the cytotoxicagent, or pharmaceutically acceptable salt thereof, has limited or nosystemic release, systemic toxicity, poor PK characteristics, orcombinations thereof. In some embodiments of the pharmaceuticalcompositions or devices described herein, the cytotoxic agent is in theform of a neutral molecule, free base, a free acid, a salt, a prodrug,or a combination thereof. In some embodiments of the pharmaceuticalcompositions or devices described herein, the cytotoxic agent isadministered in the form of an ester prodrug. In some embodimentspharmaceutical compositions or devices described herein comprise one ormore cytotoxic agent, or pharmaceutically acceptable salt thereof,prodrug or combination thereof as an immediate release agent.

In some embodiments, the controlled release composition furthercomprises an additional therapeutic agent, including an additionalcytotoxic agent, a corticosteroid, an anti-TNF agent, a chemotherapeuticagent, including additional chemotherapeutic agents that are notcytotoxic (e.g., agents that are given to treat symptoms of cancer orside effects of other chemotherapeutic agents), a collagen, agamma-globulin, an interferon, an anti-microbial agent, an antibiotic, aplatelet activator factor antagonist, a nitric oxide synthase inhibitor,or combinations thereof. In another aspect, the additional therapeuticagent is an immediate release or a controlled release agent.

In some embodiments, pharmaceutical compositions or devices describedherein are pharmaceutical compositions or devices wherein the pH of thepharmaceutical composition or device is between about 6.0 to about 7.6.

In some embodiments of the pharmaceutical compositions or devicesdescribed herein, the ratio of a polyoxyethylene-polyoxypropylenetriblock copolymer of general formula E106 P70 E106 to a thickeningagent is from about 40:1 to about 5:1. In some embodiments, thethickening agent is carboxymethyl cellulose, hydroxypropyl cellulose orhydroxypropyl methylcellulose.

In some embodiments, the otic disease or condition is cancer of the ear,Meniere's disease, sensorineural hearing loss, noise induced hearingloss, auto immune ear disease (AIED) or tinnitus.

Also provided herein is a method of treating an otic disease orcondition comprising administering to an individual in need thereof anintratympanic composition or device comprising a therapeuticallyeffective amount of cytotoxic agent, the intratympanic composition ordevice comprising substantially low degradation products of thecytotoxic agent, the intratympanic composition or device furthercomprising two or more characteristics selected from:

-   -   (i) between about 0.11% to about 10% by weight of the cytotoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate cytotoxic agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation, and    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject.

In some embodiments of the methods described herein, the cytotoxic agentis released from the composition or device for a period of at least 3days. In some embodiments of the methods described herein, the cytotoxicagent is released from the composition or device for a period of atleast 5 days. In some embodiments of the methods described herein, thecytotoxic agent is released from the composition or device for a periodof at least 10 days. In some embodiments of the method described above,the cytotoxic agent is essentially in the form of micronized particles.

In some embodiments of the methods described herein, the composition isadministered across the round window. In some embodiments of the methodsdescribed herein, the otic disease or condition is cancer of the ear,Meniere's disease, sensorineural hearing loss, noise induced hearingloss, auto immune ear disease or tinnitus.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. illustrates a comparison of non-sustained release and sustainedrelease formulations.

FIG. 2 illustrates the effect of concentration on the viscosity ofaqueous solutions of Blanose refined CMC.

FIG. 3 illustrates the effect of concentration on the viscosity ofaqueous solutions of Methocel.

FIG. 4 illustrates the anatomy of the ear

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are controlled release cytotoxic agent compositions andformulations for the treatment of autoimmune diseases of the ear,including autoimmune inner ear disease (AIED). Also provided herein arecontrolled release cytotoxic agent compositions for the treatment ofdisorders of the auris media, including otitis media. The compositionsdisclosed herein are also useful for the treatment of cancer,particularly cancer of the ear. In certain embodiments, the cytotoxicagent is an antimetabolite, an antifolate, an alkylating agent, a DNAintercalator, an anti-TNF agent, an anti-angiogenic agent, ananti-inflammatory agent, and/or an immunomodulatory agent. In certainspecific embodiments, the cytotoxic agent of the composition ismethotrexate, cyclophosphamide, or thalidomide, or a metabolite, salt,polymorph, prodrug, analogue, or derivative of methotrexate,cyclophosphamide, or thalidomide. In certain embodiments, preferredmetabolites of cyclophosphamide for incorporation into the compositionsand formulations disclosed herein are 4-hydroxycyclophosphamide,aldophosphamide, phosphoramide mustard, or combinations thereof.Compositions comprising combinations of therapeutic agents useful forthe treatment of otic disorders, including combinations of the differentcytotoxic agents, as well as combinations of cytotoxic agents with othertherapeutic agents, are also encompassed by the disclosure herein.

AIED consists of a syndrome of progressive hearing loss and/ordizziness. The origin of AIED is likely autoantibodies and/or immunecells attacking inner ear structures, but may be associated with otherautoimmune conditions, including Ankylosing spondylitis, Systemic LupusErythematosis (SLE), Sjögren's Syndrome, Cogan's disease, ulcerativecolitis, Wegener's granulomatosis, rheumatoid arthritis, scleroderma andBehçet's disease.

A few therapeutic products are available for the treatment of AIED,including certain cytotoxic agents. Particularly, the cytotoxic agentsmethotrexate and cyclophosphamide have been tested and are used forsystemic treatment of AIED. Also, thalidomide, while not currentlyadministered for the treatment of AIED, has been used to treat Behçet'sdisease, which is often associated with AIED. Despite the ability ofcytotoxic agents to treat AIED, or AIED-associated diseases, whenadministered systemically, systemic administration has significantdrawbacks.

Systemic routes via oral, intravenous or intramuscular routes arecurrently used to deliver cytotoxic agent therapeutic agents. Systemicdrug administration may create a potential inequality in drugconcentration with higher circulating levels in the serum, and lowerlevels in the target auris interna organ structures. As a result, fairlylarge amounts of drug are required to overcome this inequality in orderto deliver sufficient, therapeutically effective quantities to the innerear. In addition, systemic drug administration may increase thelikelihood of systemic toxicities and adverse side effects as a resultof the high serum amounts required to effectuate sufficient localdelivery to the target site. Systemic toxicities may also occur as aresult of liver breakdown and processing of the therapeutic agents,forming toxic metabolites that effectively erase any benefit attainedfrom the administered therapeutic.

Typical side effects of systemic administration of cytotoxic agents,e.g., methotrexate, cyclophosphamide, and thalidomide, include anemia,neutropenia, bruising, nausea, dermatitis, hepatitis, pulmonaryfibrosis, teratogenicity, peripheral neuropathy, fatigue, constipation,deep vein thrombosis, pulmonary edema, atelectasis, aspirationpneumonia, hypotension, bone marrow suppression, diarrhea, darkening ofskin and nails, alopecia, changes in hair color and texture, lethargy,hemorrhagic cystitis, carcinoma, mouth sores, and decreased immunity.

In addition, localized treatment of the auris interna also affords theuse of previously undesired therapeutic agents, including agents withpoor pK profiles, poor uptake, low systemic release and/or toxicityissues. Because of the localized targeting of the cytotoxic agentformulations and compositions, as well as the biological blood barrierpresent in the auris interna, the risk of adverse effects will bereduced as a result of treatment with previously characterized toxic orineffective cytotoxic agents. Accordingly, also contemplated within thescope of the embodiments herein is the use of cytotoxic agents in thetreatment of otic disorders (e.g., cancer) that have been previouslyrejected by practitioners because of adverse effects or ineffectivenessof the cytotoxic agents (e.g., thalidomide).

Treatment of cancer, particularly cancer of the ear, by systemicadministration of conventional anti-cancer agents, e.g., cytotoxicagents, suffers from the same drawbacks as treatment of AIED with theagents. For example, systemic administration of cytotoxic agents totreat cancer of the ear results in higher systemic levels of the agentsthan levels in the target auris media and auris interna organstructures. Furthermore, the increased doses of the agents that arerequired to achieve the desired therapeutic levels in the target earstructures result in increased levels of the adverse side effectsdiscussed above, thus limiting the therapeutic potential for systemicadministration of these agents to treat localized cancers, such ascancer of the ear.

Also included within the embodiments disclosed herein is the use ofadditional auris media and/or auris interna-acceptable agents incombination with the cytotoxic agent formulations and compositionsdisclosed herein. When used, such agents assist in the treatment ofhearing or equilibrium loss or dysfunction resulting from an autoimmunedisorder, including vertigo, tinnitus, hearing loss, balance disorders,infections, inflammatory response or combinations thereof. Accordingly,agents that ameliorate or reduce the effects of vertigo, tinnitus,hearing loss, balance disorders, infections, inflammatory response orcombinations thereof are also contemplated to be used in combinationwith the cytotoxic agent(s), including additional cytotoxic agents,steroids, anti-emetic agents, corticosteroids, chemotherapeutic agents,including additional chemotherapeutic agents that may not be cytotoxic(e.g., agents that are given to treat symptoms of cancer or side effectsof other chemotherapeutic agents); treatment with collagen, gammaglobulin, interferons, copaxone, central nervous system agents,antibiotics, platelet-activating factor antagonists, nitric oxidesynthase inhibitors and combinations thereof.

Further included in the embodiments disclosed herein is the use of otherauris media and/or auris interna-acceptable agents in combination withthe cytotoxic agent formulations and compositions for the treatment ofcancer, particularly cancer of the ear, or symptoms that result from thecancer, such as, in the case of cancer of the ear, otitis media orexterna, facial palsy, and vertigo. Agents used in combination with theformulations and compositions disclosed herein for the treatment ofcancer and conditions and/or symptoms associated with the cancer,corticosteroids such as prednisone, and antibiotics such as amoxicillin,clavulanate acid, trimethoprim-sulfamethoxazole, cefuroxime,clarithromycin and azithromycin and other cephalosporins, macrolides,penicillins or sulfonamides.

To overcome the toxic and attendant undesired side effects of systemicdelivery of cytotoxic agents (which are generally understood to be toxicto cells), disclosed herein are methods and compositions for localdelivery of cytotoxic agents to auris media and/or auris internastructures. Access to, for example, the vestibular and cochlearapparatus will occur through the auris media or auris interna, includingthe round window membrane, the oval window/stapes footplate, the annularligament and through the otic capsule/temporal bone. In further oralternative embodiments, the auris controlled-release formulations arecapable of being administered on or near the round window membrane viaintratympanic injection. In other embodiments, the auris controlledrelease formulations are administered on or near the round window or thecrista fenestrae cochleae through entry via a post-auricular incisionand surgical manipulation into or near the round window or the cristafenestrae cochleae area. Alternatively, the auris controlled releaseformulation is applied via syringe and needle, wherein the needle isinserted through the tympanic membrane and guided to the area of theround window or crista fenestrae cochleae.

Accordingly, provided herein are controlled release cytotoxic agentformulations and compositions to locally treat auris media and/or aurisinterna structures, thereby avoiding side effects as a result ofsystemic administration of the cytotoxic agents. The locally appliedcytotoxic agent formulations and compositions are compatible with aurismedia and/or auris interna structures, and are administered eitherdirectly to the desired auris media and/or auris interna structure, e.g.the cochlear region or the tympanic cavity, or administered to astructure in direct communication with areas of the auris interna,including but not limited to the round window membrane, the cristafenestrae cochleae or the oval window membrane. By specificallytargeting the auris media or auris interna structures, adverse sideeffects as a result of systemic treatment are avoided. Moreover, byproviding a controlled release cytotoxic agent formulation orcomposition to treat otic disorders, a constant and/or extended sourceof cytotoxic agent is provided to the individual or patient sufferingfrom an otic disorder, reducing or eliminating the variability oftreatment.

Intratympanic injection of therapeutic agents is the technique ofinjecting a therapeutic agent behind the tympanic membrane into theauris media and/or auris interna. Despite early success with thistechnique (Schuknecht, Laryngoscope (1956) 66, 859-870) some challengesdo remain. For example, access to the round window membrane, the site ofdrug absorption into the auris interna, can be challenging.

However, intra-tympanic injections create several unrecognized problemsnot addressed by currently available treatment regimens, such aschanging the osmolarity and pH of the perilymph and endolymph, andintroducing pathogens and endotoxins that directly or indirectly damageinner ear structures. One of the reasons the art may not have recognizedthese problems is that there are no approved intra-tympaniccompositions: the inner ear provides sui generis formulation challenges.Thus, compositions developed for other parts of the body have little tono relevance for an intra-tympanic composition.

There is no guidance in the prior art regarding requirements (e.g.,level of sterility, pH, osmolarity) for otic formulations that aresuitable for administration to humans. There is wide anatomicaldisparity between the ears of animals across species. A consequence ofthe inter-species differences in auditory structures is that animalmodels of inner ear disease are often unreliable as a tool for testingtherapeutics that are being developed for clinical approval.

Provided herein are otic formulations that meet stringent criteria forpH, osmolarity, ionic balance, sterility, endotoxin and/or pyrogenlevels. The auris compositions described herein are compatible with themicroenvironment of the inner ear (e.g., the perilymph) and are suitablefor administration to humans. In some embodiments, the formulationsdescribed herein comprise dyes and aid visualization of the administeredcompositions obviating the need for invasive procedures (e.g., removalof perilymph) during preclinical and/or clinical development ofintratympanic therapeutics.

Provided herein are controlled release cytotoxic agent formulations andcompositions to locally treat targeted auris structures, therebyavoiding side effects as a result of systemic administration of thecytotoxic agent formulations and compositions. The locally appliedcytotoxic agent formulations and compositions and devices are compatiblewith the targeted auris structures, and administered either directly tothe desired targeted auris structure, e.g. the cochlear region, thetympanic cavity or the external ear, or administered to a structure indirect communication with areas of the auris interna, including but notlimited to the round window membrane, the crista fenestrae cochleae orthe oval window membrane. By specifically targeting an auris structure,adverse side effects as a result of systemic treatment are avoided.Moreover, clinical studies have shown the benefit of having long termexposure of drug to the perilymph of the cochlea, for example withimproved clinical efficacy of sudden hearing loss when the therapeuticagent is given on multiple occasions. Thus, by providing a controlledrelease cytotoxic agent formulation or composition to treat oticdisorders, a constant, and/or extended source of cytotoxic agent isprovided to the individual or patient suffering from an otic disorder,reducing or eliminating variabilities in treatment. Accordingly, oneembodiment disclosed herein is to provide a composition that enables atleast one cytotoxic agent to be released in therapeutically effectivedoses either at variable or constant rates such as to ensure acontinuous release of the at least one agent. In some embodiments, thecytotoxic agents disclosed herein are administered as an immediaterelease formulation or composition. In other embodiments, the cytotoxicagents are administered as a sustained release formulation, releasedeither continuously, variably or in a pulsatile manner, or variantsthereof. In still other embodiments, cytotoxic agent formulation isadministered as both an immediate release and sustained releaseformulation, released either continuously, variably or in a pulsatilemanner, or variants thereof. The release is optionally dependent onenvironmental or physiological conditions, for example, the externalionic environment (see, e.g. Oros® release system, Johnson & Johnson).

In addition, the auris-acceptable controlled-release cytotoxic agentformulations and treatments described herein are provided to the targetear region of the individual in need, including the inner ear, and theindividual in need is additionally administered an oral dose ofcytotoxic agent. In some embodiments, the oral dose of cytotoxic agentis administered prior to administration of the auris-acceptablecontrolled-release cytotoxic agent formulation, and then the oral doseis tapered off over the period of time that the auris-acceptablecontrolled-release cytotoxic agent formulation is provided.Alternatively, the oral dose of cytotoxic agent is administered duringadministration of the auris-acceptable controlled-release cytotoxicagent formulation, and then the oral dose is tapered off over the periodof time that the auris-acceptable controlled-release cytotoxic agentformulation is provided. Alternatively, the oral dose of cytotoxic agentis administered after administration of the auris-acceptablecontrolled-release cytotoxic agent formulation has been initiated, andthen the oral dose is tapered off over the period of time that theauris-acceptable controlled-release cytotoxic agent formulation isprovided.

In addition, the cytotoxic agent pharmaceutical compositions orformulations or devices included herein also include carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressure,and/or buffers. Such carriers, adjuvants, and other excipients will becompatible with the environment in the targeted auris structure(s).Accordingly, specifically contemplated are carriers, adjuvants andexcipients that lack ototoxicity or are minimally ototoxic in order toallow effective treatment of the otic disorders contemplated herein withminimal side effects in the targeted regions or areas. To preventototoxicity, cytotoxic agent pharmaceutical compositions or formulationsor devices disclosed herein are optionally targeted to distinct regionsof the targeted auris structures, including but not limited to thetympanic cavity, vestibular bony and membranous labyrinths, cochlearbony and membranous labyrinths and other anatomical or physiologicalstructures located within the auris interna.

CERTAIN DEFINITIONS

The term “auris-acceptable” with respect to a formulation, compositionor ingredient, as used herein, includes having no persistent detrimentaleffect on the auris interna (or inner ear) of the subject being treated.By “auris-pharmaceutically acceptable,” as used herein, refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound in reference to theauris interna (or inner ear), and is relatively or is reduced intoxicity to the auris interna (or inner ear), i.e., the material isadministered to an individual without causing undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the composition in which it is contained.

As used herein, amelioration or lessening of the symptoms of aparticular otic disease, disorder or condition by administration of aparticular compound or pharmaceutical composition refers to any decreaseof severity, delay in onset, slowing of progression, or shortening ofduration, whether permanent or temporary, lasting or transient that isattributed to or associated with administration of the compound orcomposition.

As used herein, the term “cytotoxic agent” refers to compounds that arecytotoxic (i.e., toxic to a cell) effective for the treatment of oticdisorders, e.g., autoimmune diseases of the ear and cancer of the ear,and are suitable for use in the formulations disclosed herein.

The phrase “cytotoxic small molecule” refers to cytotoxic compounds thatare of relatively low molecular weight, e.g., less than 1,000, or lessthan 600-700, or between 300-700 molecular weight, that are effectivefor the treatment of otic disorders, e.g., autoimmune diseases of theear and cancer of the ear, and are suitable for use in the formulationsdisclosed herein. Suitable “cytotoxic small molecules” includemethotrexate, cyclophosphamide, and thalidomide, as well as metabolites,salts, polymorphs, prodrugs, analogues, and derivatives of methotrexate,cyclophosphamide, and thalidomide. In certain embodiments, preferredcytotoxic small molecules are the pharmaceutically active metabolites ofcytotoxic agents. For example, in the case of cyclophosphamide,preferred metabolites are pharmaceutically active metabolites ofcyclophosphamide, including but not limited to4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard, andcombinations thereof.

“Antioxidants” are auris-pharmaceutically acceptable antioxidants, andinclude, for example, butylated hydroxytoluene (BHT), sodium ascorbate,ascorbic acid, sodium metabisulfite and tocopherol. In certainembodiments, antioxidants enhance chemical stability where required.Antioxidants are also used to counteract the ototoxic effects of certaintherapeutic agents, including agents that are used in combination withthe cytotoxic agents disclosed herein.

“Auris interna” refers to the inner ear, including the cochlea and thevestibular labyrinth, and the round window that connects the cochleawith the middle ear.

“Auris-interna bioavailability” refers to the percentage of theadministered dose of compounds disclosed herein that becomes availablein the inner ear of the animal or human being studied.

“Auris media” refers to the middle ear, including the tympanic cavity,auditory ossicles and oval window, which connects the middle ear withthe inner ear.

“Blood plasma concentration” refers to the concentration of compoundsprovided herein in the plasma component of blood of a subject.

“Carrier materials” are excipients that are compatible with thecytotoxic agent, the auris interna and the release profile properties ofthe auris-acceptable pharmaceutical formulations. Such carrier materialsinclude, e.g., binders, suspending agents, disintegration agents,filling agents, surfactants, solubilizers, stabilizers, lubricants,wetting agents, diluents, and the like. “Auris-pharmaceuticallycompatible carrier materials” include, but are not limited to, acacia,gelatin, colloidal silicon dioxide, calcium glycerophosphate, calciumlactate, maltodextrin, glycerine, magnesium silicate,polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodiumcaseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodiumchloride, tricalcium phosphate, dipotassium phosphate, cellulose andcellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like.

The term “diluent” refers to chemical compounds that are used to dilutethe cytotoxic agent prior to delivery and which are compatible with theauris interna.

“Dispersing agents,” and/or “viscosity modulating agents” are materialsthat control the diffusion and homogeneity of the cytotoxic agentthrough liquid media. Examples of diffusion facilitators/dispersingagents include but are not limited to hydrophilic polymers,electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP;commercially known as Plasdone®, and the carbohydrate-based dispersingagents such as, for example, hydroxypropyl celluloses (e.g., HPC,HPC-SL, and HPC-L), hydroxypropyl methylcelluloses (e.g., HPMC K100,HPMC K4M, HPMC K15M, and HPMC K100M), carboxymethylcellulose sodium,methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcelluloseacetate stearate (HPMCAS), noncrystalline cellulose, magnesium aluminumsilicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolhas a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose are also be used as dispersing agents. Dispersingagents useful in liposomal dispersions and self-emulsifying dispersionsof the cytotoxic agents disclosed herein are dimyristoyl phosphatidylcholine, natural phosphatidyl choline from eggs, natural phosphatidylglycerol from eggs, cholesterol and isopropyl myristate.

“Drug absorption” or “absorption” refers to the process of movement ofthe cytotoxic agents from the localized site of administration, by wayof example only, the round window membrane of the inner ear, and acrossa barrier (the round window membranes, as described below) into theauris interna or inner ear structures. The terms “co-administration” orthe like, as used herein, are meant to encompass administration of thecytotoxic agents to a single patient, and are intended to includetreatment regimens in which the cytotoxic agents are administered by thesame or different route of administration or at the same or differenttime.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of the cytotoxic agent beingadministered that would be expected to relieve to some extent one ormore of the symptoms of the disease or condition being treated. Forexample, the result of administration of the cytotoxic agents disclosedherein is reduction and/or alleviation of the signs, symptoms, or causesof AIED in the inner ear. For example, an “effective amount” fortherapeutic use is the amount of the cytotoxic agent, including aformulation as disclosed herein required to provide a decrease oramelioration in disease symptoms without undue adverse side effects. Theterm “therapeutically effective amount” includes, for example, aprophylactically effective amount. An “effective amount” of a cytotoxicagent composition disclosed herein is an amount effective to achieve adesired pharmacologic effect or therapeutic improvement without undueadverse side effects. It is understood that “an effective amount” or “atherapeutically effective amount” varies, in some embodiments, fromsubject to subject, due to variation in metabolism of the compoundadministered, age, weight, general condition of the subject, thecondition being treated, the severity of the condition being treated,and the judgment of the prescribing physician. It is also understoodthat “an effective amount” in an extended release dosing format maydiffer from “an effective amount” in an immediate release dosing formatbased upon pharmacokinetic and pharmacodynamic considerations.

The terms “enhance” or “enhancing” refers to an increase or prolongationof either the potency or duration of a desired effect of the cytotoxicagents or a diminution of any adverse symptomatology that is consequentupon the administration of the cytotoxic agent. Thus, in regard toenhancing the effect of the cytotoxic agents disclosed herein, the term“enhancing” refers to the ability to increase or prolong, either inpotency or duration, the effect of other therapeutic agents that areused in combination with the cytotoxic agents disclosed herein. An“enhancing-effective amount,” as used herein, refers to an amount ofcytotoxic agent or other therapeutic agent that is adequate to enhancethe effect of another therapeutic agent or cytotoxic agent in a desiredsystem. When used in a patient, amounts effective for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician.

The term “inhibiting” includes preventing, slowing, or reversing thedevelopment of a condition, for example, or advancement of a conditionin a patient necessitating treatment.

The terms “kit” and “article of manufacture” are used as synonyms.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at the desiredsite within the auris media and/or auris interna.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at the desiredsite within the auris media and/or auris interna.

In prophylactic applications, compositions comprising the cytotoxicagents described herein are administered to a patient susceptible to orotherwise at risk of a particular disease, disorder or condition, forexample, AIED, or patients that are suffering from diseases associatedwith AIED, including by way of example only, Ankylosing spondylitis,Systemic Lupus Erythematosus (SLE), Sjögren's Syndrome, Cogan's disease,ulcerative colitis, Wegener's granulomatosis, inflammatory boweldisease, rheumatoid arthritis, scleroderma and Behçet's disease. Inother embodiments for prophylactic application, compositions comprisingthe cytotoxic agents described herein are administered to a patientsusceptible to or otherwise at risk of cancer, particularly cancer ofthe ear. Such an amount is defined to be a “prophylactically effectiveamount or dose.” In this use, the precise amounts also depend on thepatient's state of health, weight, and the like.

As used herein, a “pharmaceutical device” includes any compositiondescribed herein that, upon administration to an ear, provides areservoir for extended release of an active agent described herein.

The term “substantially low degradation products” means less than 5% byweight of the active agent are degradation products of the active agent.In further embodiments, the term means less than 3% by weight of theactive agent are degradation products of the active agent. In yetfurther embodiments, the term means less than 2% by weight of the activeagent are degradation products of the active agent. In furtherembodiments, the term means less than 1% by weight of the active agentare degradation products of the active agent. In some embodiments, anyindividual impurity (e.g., metal impurity, degradation products ofactive agent and/or excipients, or the like) present in a formulationdescribed herein is less than 5%, less than 2%, or less than 1% byweight of the active agent. In some embodiments, the formulation doesnot contain precipitate during storage or change in color aftermanufacturing and storage.

A “prodrug” refers to a cytotoxic agent that is converted into theparent drug in vivo. In certain embodiments, a prodrug is enzymaticallymetabolized by one or more steps or processes to the biologically,pharmaceutically or therapeutically active form of the compound. Toproduce a prodrug, a pharmaceutically active compound is modified suchthat the active compound will be regenerated upon in vivoadministration. In one embodiment, the prodrug is designed to alter themetabolic stability or the transport characteristics of a drug, to maskside effects or toxicity, or to alter other characteristics orproperties of a drug. Compounds provided herein, in some embodiments,are derivatized into suitable prodrugs.

“Solubilizers” refer to auris-acceptable compounds such as triacetin,triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate,sodium doccusate, vitamin E TPGS, dimethylacetamide,N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone,hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol,n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethyleneglycol 200-600, glycofurol, transcutol, propylene glycol, and dimethylisosorbide and the like that assist or increase the solubility of thecytotoxic agents disclosed herein.

“Stabilizers” refers to compounds such as any antioxidation agents,buffers, acids, preservatives and the like that are compatible with theenvironment of the auris interna. Stabilizers include but are notlimited to agents that will do any of (1) improve the compatibility ofexcipients with a container, or a delivery system, including a syringeor a glass bottle, (2) improve the stability of a component of thecomposition, or (3) improve formulation stability.

“Steady state,” as used herein, is when the amount of drug administeredto the auris interna is equal to the amount of drug eliminated withinone dosing interval resulting in a plateau or constant levels of drugexposure within the targeted structure.

As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human or non-human. The terms patient and subjectmay be used interchangeably.

“Surfactants” refer to compounds that are auris-acceptable, such assodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin,vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitanmonooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate,copolymers of ethylene oxide and propylene oxide, e.g., Pluronic®(BASF), and the like. Some other surfactants include polyoxyethylenefatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60)hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenylethers, e.g., octoxynol 10, octoxynol 40. In some embodiments,surfactants are included to enhance physical stability or for otherpurposes.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition, for exampleAIED and/or cancer of the ear, symptoms, preventing additional symptoms,ameliorating or preventing the underlying metabolic causes of symptoms,inhibiting the disease or condition, e.g., arresting the development ofthe disease or condition, relieving the disease or condition, causingregression of the disease or condition, relieving a condition caused bythe disease or condition, or stopping the symptoms of the disease orcondition either prophylactically and/or therapeutically.

Other objects, features, and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only.

Anatomy of the Ear

As shown FIG. 4, the outer ear is the external portion of the organ andis composed of the pinna (auricle), the auditory canal (externalauditory meatus) and the outward facing portion of the tympanicmembrane, also known as the ear drum. The pinna, which is the fleshypart of the external ear that is visible on the side of the head,collects sound waves and directs them toward the auditory canal. Thus,the function of the outer ear, in part, is to collect and direct soundwaves towards the tympanic membrane and the middle ear.

The middle ear is an air-filled cavity, called the tympanic cavity,behind the tympanic membrane. The tympanic membrane, also known as theear drum, is a thin membrane that separates the external ear from themiddle ear. The middle ear lies within the temporal bone, and includeswithin this space the three ear bones (auditory ossicles): the malleus,the incus and the stapes. The auditory ossicles are linked together viatiny ligaments, which form a bridge across the space of the tympaniccavity. The malleus, which is attached to the tympanic membrane at oneend, is linked to the incus at its anterior end, which in turn is linkedto the stapes. The stapes is attached to the oval window, one of twowindows located within the tympanic cavity. A fibrous tissue layer,known as the annular ligament connects the stapes to the oval window.Sound waves from the outer ear first cause the tympanic membrane tovibrate. The vibration is transmitted across to the cochlea through theauditory ossicles and oval window, which transfers the motion to thefluids in the auris interna. Thus, the auditory ossicles are arranged toprovide a mechanical linkage between the tympanic membrane and the ovalwindow of the fluid-filled auris interna, where sound is transformed andtransduced to the auris interna for further processing. Stiffness,rigidity or loss of movement of the auditory ossicles, tympanic membraneor oval window leads to hearing loss, e.g. otosclerosis, or rigidity ofthe stapes bone.

The tympanic cavity also connects to the throat via the eustachian tube.The eustachian tube provides the ability to equalize the pressurebetween the outside air and the middle ear cavity. The round window, acomponent of the auris interna but which is also accessible within thetympanic cavity, opens into the cochlea of the auris interna. The roundwindow is covered by round window membrane, which consists of threelayers: an external or mucous layer, an intermediate or fibrous layer,and an internal membrane, which communicates directly with the cochlearfluid. The round window, therefore, has direct communication with theauris interna via the internal membrane.

Movements in the oval and round window are interconnected, i.e. as thestapes bone transmits movement from the tympanic membrane to the ovalwindow to move inward against the auris interna fluid, the round window(round window membrane) is correspondingly pushed out and away from thecochlear fluid. This movement of the round window allows movement offluid within the cochlea, which leads in turn to movement of thecochlear inner hair cells, allowing hearing signals to be transduced.Stiffness and rigidity in round window membrane leads to hearing lossbecause of the lack of ability of movement in the cochlear fluid. Recentstudies have focused on implanting mechanical transducers onto the roundwindow, which bypasses the normal conductive pathway through the ovalwindow and provides amplified input into the cochlear chamber.

Auditory signal transduction takes place in the auris interna. Thefluid-filled auris interna, or inner ear, consists of two majorcomponents: the cochlear and the vestibular apparatus. The auris internais located in part within the osseous or bony labyrinth, an intricateseries of passages in the temporal bone of the skull. The vestibularapparatus is the organ of balance and consists of the threesemi-circular canals and the vestibule. The three semi-circular canalsare arranged relative to each other such that movement of the head alongthe three orthogonal planes in space can be detected by the movement ofthe fluid and subsequent signal processing by the sensory organs of thesemi-circular canals, called the crista ampullaris. The cristaampullaris contains hair cells and supporting cells, and is covered by adome-shaped gelatinous mass called the cupula. The hairs of the haircells are embedded in the cupula. The semi-circular canals detectdynamic equilibrium, the equilibrium of rotational or angular movements.

When the head turns rapidly, the semicircular canals move with the head,but endolymph fluid located in the membranous semi-circular canals tendsto remain stationary. The endolymph fluid pushes against the cupula,which tilts to one side. As the cupula tilts, it bends some of the hairson the hair cells of the crista ampullaris, which triggers a sensoryimpulse. Because each semicircular canal is located in a differentplane, the corresponding crista ampullaris of each semi-circular canalresponds differently to the same movement of the head. This creates amosaic of impulses that are transmitted to the central nervous system onthe vestibular branch of the vestibulocochlear nerve. The centralnervous system interprets this information and initiates the appropriateresponses to maintain balance. Of importance in the central nervoussystem is the cerebellum, which mediates the sense of balance andequilibrium.

The vestibule is the central portion of the auris interna and containsmechanoreceptors bearing hair cells that ascertain static equilibrium,or the position of the head relative to gravity. Static equilibriumplays a role when the head is motionless or moving in a straight line.The membranous labyrinth in the vestibule is divided into two sac-likestructures, the utricle and the saccule. Each structure in turn containsa small structure called a macula, which is responsible for maintenanceof static equilibrium. The macula consists of sensory hair cells, whichare embedded in a gelatinous mass (similar to the cupula) that coversthe macula. Grains of calcium carbonate, called otoliths, are embeddedon the surface of the gelatinous layer.

When the head is in an upright position, the hairs are straight alongthe macula. When the head tilts, the gelatinous mass and otoliths tiltscorrespondingly, bending some of the hairs on the hair cells of themacula. This bending action initiates a signal impulse to the centralnervous system, which travels via the vestibular branch of thevestibulocochlear nerve, which in turn relays motor impulses to theappropriate muscles to maintain balance.

The cochlea is the portion of the auris interna related to hearing. Thecochlea is a tapered tube-like structure which is coiled into a shaperesembling a snail. The inside of the cochlea is divided into threeregions, which is further defined by the position of the vestibularmembrane and the basilar membrane. The portion above the vestibularmembrane is the scala vestibuli, which extends from the oval window tothe apex of the cochlea and contains perilymph fluid, an aqueous liquidlow in potassium and high in sodium content. The basilar membranedefines the scala tympani region, which extends from the apex of thecochlea to the round window and also contains perilymph. The basilarmembrane contains thousands of stiff fibers, which gradually increase inlength from the round window to the apex of the cochlea. The fibers ofthe basement membrane vibrate when activated by sound. In between thescala vestibuli and the scala tympani is the cochlear duct, which endsas a closed sac at the apex of the cochlea. The cochlear duct containsendolymph fluid, which is similar to cerebrospinal fluid and is high inpotassium.

The organ of Corti, the sensory organ for hearing, is located on thebasilar membrane and extends upward into the cochlear duct. The organ ofCorti contains hair cells, which have hairlike projections that extendfrom their free surface, and contacts a gelatinous surface called thetectorial membrane. Although hair cells have no axons, they aresurrounded by sensory nerve fibers that form the cochlear branch of thevestibulocochlear nerve (cranial nerve VIII).

As discussed, the oval window, also known as the elliptical windowcommunicates with the stapes to relay sound waves that vibrate from thetympanic membrane. Vibrations transferred to the oval window increasespressure inside the fluid-filled cochlea via the perilymph and scalavestibuli/scala tympani, which in turn causes the round window membraneto expand in response. The concerted inward pressing of the ovalwindow/outward expansion of the round window allows for the movement offluid within the cochlea without a change of intra-cochlear pressure.However, as vibrations travel through the perilymph in the scalavestibuli, they create corresponding oscillations in the vestibularmembrane. These corresponding oscillations travel through the endolymphof the cochlear duct, and transfer to the basilar membrane. When thebasilar membrane oscillates, or moves up and down, the organ of Cortimoves along with it. The hair cell receptors in the Organ of Corti thenmove against the tectorial membrane, causing a mechanical deformation inthe tectorial membrane. This mechanical deformation initiates the nerveimpulse which travels via the vestibulocochlear nerve to the centralnervous system, mechanically transmitting the sound wave received intosignals that are subsequently processed by the central nervous system.

Diseases

Otic disorders, including auris interna and auris media disorders,produce symptoms which include but are not limited to hearing loss,nystagmus, vertigo, tinnitus, inflammation, swelling, infection andcongestion. These disorders may have many causes, such as infection,injury, inflammation, tumors and adverse response to drugs or otherchemical agents. Several causes of hearing and/or equilibrium impairmentor inflammation may be attributed to an autoimmune disorder and/or acytokine-mediated inflammatory response.

Autoimmune Inner Ear Disease

Autoimmune inner ear disease (AIED) is one of the few reversible causesof sensorineural hearing loss. It is a disorder appearing in both adultsand children that often involves a bilateral disturbance of the audioand vestibular functions of the auris interna. In many cases, AIEDoccurs without systemic autoimmune symptoms, but up to one-third ofpatients also suffer from a systemic autoimmune illness, such asinflammatory bowel disease, rheumatoid arthritis, Ankylosingspondylitis, Systemic Lupus Erythematosus (SLE), Sjögren's Syndrome,Cogan's disease, ulcerative colitis, Wegener's granulomatosis andscleroderma. Behçet's disease, a multisystem disease, also commonly hasaudiovestibular problems. There is some evidence for food-relatedallergies as a cause for cochlear and vestibular autoimmunity, but thereis presently no agreement as to its importance in the aetiology of thedisease. A classification scheme for AIED has been developed (Harris andKeithley, (2002) Autoimmune inner ear disease, in OtorhinolaryngologyHead and Neck Surgery. 91, 18-32).

The immune system normally performs a crucial role in protecting theinner ear from invasive pathogens such as bacteria and viruses. However,in AIED the immune system itself begins to damage the delicate inner eartissues. The inner ear is fully capable of mounting a localized immuneresponse to foreign antigens. When a foreign antigen enters the innerear, it is first processed by immunocompetent cells which reside in andaround the endolymphatic sac. Once the foreign antigen has beenprocessed by these immunocompetent cells, these cells secrete variouscytokines which modulate the immune response of the inner ear. Oneresult of this cytokine release is to facilitate the influx ofinflammatory cells, which are recruited from the systemic circulation.These systemic inflammatory cells enter the cochlea via diapedesisthrough the spiral modiolar vein and its tributaries, and begin toparticipate in antigen uptake and deregulation just as it occurs inother parts of the body. Interleukin 1 (IL-1) plays an important role inmodulating the innate (nonspecific) immune response and is a knownactivator of resting T helper cells and B-cells. T helper cells, onceactivated by IL-1, produce IL-2. IL-2 secretion results indifferentiation of pluripotent T-cells into helper, cytotoxic andsuppressor T-cell subtypes. IL-2 also assists T helper cells in theactivation of B lymphocytes and probably plays a pivotal role in theimmunoregulation of the immune response of the vestibular and cochlearregions. IL-2 is within the perilymph of the auris interna as early as 6h after antigen challenge with peak levels at 18 h after antigenchallenge. The perilymphatic levels of IL-2 then dissipate, and it is nolonger present within the perilymph at 120 hours post antigen challenge.

Both IL-1β and tumor necrosis factor-α (TNF-α) may play a key role inthe initiation and amplification of the immune response. IL-1β isexpressed by the fibrocytes of the spiral ligament in the presence oftrauma such as surgical trauma or acoustic trauma in a nonspecificresponse. TNF-α is expressed either by infiltrating systemic cells or byresident cells contained within the endolymphatic sac in the presence ofantigen. TNF-α is released as part of the adaptive (specific) immuneresponse in animal models. When antigen is injected into the aurisinterna of mice, IL-1β and TNF-α are both expressed and a vigorousimmune response occurs. However, when antigen is introduced to the aurisinterna via the cerebral spinal fluid in the absence of trauma, onlyTNF-α is expressed and the immune response in minimal. Importantly,cochlear trauma in isolation also results in a minimal immune response.These results suggest that both the nonspecific and specific componentsof the immune response act in concert in the auris interna to achieve amaximal response.

Thus, if the cochlea is traumatized and an antigen is injected (or inthe case of autoimmune disease, the patient has immune cells directedagainst inner ear antigens), both the nonspecific and the specificimmune responses can be activated simultaneously. This results in theconcurrent production of IL-1β as well as TNF-α which causes a greatlyamplified level of inflammation leading to substantial damage to theauris interna.

Accordingly, an embodiment of the instant invention is the treatment ofAIED by administering cytotoxic agent compositions and formulations thathave anti-inflammatory activity and/or interfere with the nonspecificand specific immune responses. Particularly, the cytotoxic agentsmethotrexate and cyclophosphamide have been tested and are used forsystemic treatment of AIED, with its attendant systemic side effects.Also, thalidomide, while not currently administered for the treatment ofAIED, has been used to treat Behçet's disease, which is often associatedwith AIED. Thus, in certain embodiments, methotrexate, cyclophosphamide,and thalidomide are suitable for use in the compositions andformulations disclosed herein for the treatment of AIED. Also suitablefor use in the compositions and formulations disclosed herein for thetreatment of AIED are metabolites, salts, polymorphs, prodrugs,analogues, and derivatives of methotrexate, cyclophosphamide, andthalidomide that retain the ability of the parent cytotoxic agents totreat AIED. Preferred metabolites of cyclophosphamide in certainembodiments are active metabolites of cyclophosphamide, including butnot limited to 4-hydroxycyclophosphamide, aldophosphamide, phosphoramidemustard, and combinations thereof.

Another embodiment is the treatment of autoimmune otic disorders,including AIED, with the cytotoxic agent compositions and formulationsdisclosed herein in conjunction with other pharmaceutical agents usefulfor treating the same conditions or symptoms of the same conditions,including steroids, chemotherapeutic agents, collagen, gamma globulininfusion, or other immune modulating drugs. Steroids include, e.g.,prednisone or decadron. Chemotherapeutic agents include, e.g.,cisplatin, azathiaprine, actinomycin, bleomycin, carboplatin andvincristine. Plasmapheresis procedures are optionally used. Treatmentwith oral collagen, gamma globulin infusions, or other immune modulatingdrugs (e.g. beta-interferon, alpha-interferon or copaxone) is alsooptionally used in combination with the cytotoxic agent compositions orformulations disclosed herein. The additional pharmaceutical agents areoptionally administered together with the controlled releasecompositions and formulations disclosed herein, or through other modesof administration, e.g., orally, by injection, topically administered,nasally administered or through any other means. The additionalpharmaceutical agents are optionally co-administered, or administered atdifferent time periods.

Inflammatory Disorders of the Auris Media

Otitis media (OM), which includes acute otitis media (AOM), otitis mediawith effusion (OME) and chronic otitis media as examples, is a conditionaffecting both adults and children. OM susceptibility is multifactorialand complex, including environmental, microbial and host factors.Bacterial infection accounts for a large percentage of OM cases, withmore than 40% of cases attributed to Streptococcus pneumoniae infection.However, viral causes, as well as other microbial agents, may alsoaccount for OM conditions.

Regardless of the causative agent, increases in cytokine production,including interleukins and TNF, have been observed in the effluent mediaof individuals afflicted with OM. IL-1β, IL-6 and TNF α are acute-phasecytokines that promote acute inflammatory response after infection withviruses and bacteria. Genetic studies supports this link betweencytokines and OM by demonstrating a correlation in the occurrence ofTNF-α SNP (single-nucleotide polymorphisms) and an increasedsusceptibility for OM in pediatric patients suffering from AOM and witha subsequent need for placement of tympanostomy tubes. (Patel et al.Pediatrics (2006) 118:2273-2279). In animal models of OM induced withpneumococci innoculations, TNF-α and interleukins levels were found toincrease in early developmental phase of OM, with TNF-α levels steadilyincreasing 72 hours after innoculation. Moreover, higher TNF-α levelshave been associated with a history of multiple tympanostomy tubeplacements, indicating a role for TNF-α in chronic OM cases. Finally,direct injection of TNF-α and interleukins has been shown to inducemiddle ear inflammation in a guinea pig model. These studies support therole that cytokines may play in the origin and maintenance of OM in theauris media.

Because OM can be caused by a virus, bacteria or both, it is oftendifficult to identify the exact cause and thus the most appropriatetreatment. Treatment options of OM in the auris media include treatmentwith antibiotics, such as amoxicillin, clavulanate acid,trimethoprim-sulfamethoxazole, cefuroxime, clarithromycin andazithromycin and other cephalosporins, macrolides, penicillins orsulfonamides. Surgical intervention is also available, including amyringotomy, an operation to insert a tympanostomy tube through thetympanic membrane and into the patient's middle ear to drain the fluidand balance the pressure between the outer and middle ear. Antipyreticsand analgesics, including benzocaine, ibuprofen and acetaminophen, mayalso be prescribed to treat accompanying fever or pain effects.Pre-treatment with TNF-α inhibitors in experimental lipopolysaccharide(LPS)-induced OM animal models has been shown to suppress development ofOM, suggesting a role in the treatment of OM or OME. In addition,treatment of such conditions include use of TNF-α inhibitors incombination with other inflammatory response mediators, includingplatelet activating factor antagonists, nitric oxide synthase inhibitorsand histamine antagonists.

As discussed above, methotrexate, cyclophosphamide, and thalidomide areall cytotoxic small molecule agents that are systemically administeredto treat AIED. Thus, the compounds are useful in the compositions andformulations disclosed herein for the treatment of inflammatorydisorders of the auris media, including OM, by having a directanti-inflammatory effect, particularly by interfering with TNF activity.In other embodiments, metabolites, salts, polymorphs, prodrugs,analogues, and derivatives of methotrexate, cyclophosphamide, andthalidomide that retain the ability of the parent cytotoxic agents totreat inflammatory disorders of the auris media, including OM, areuseful in the formulations disclosed herein for the treatment ofinflammatory disorders of the auris media, including OM. In certainembodiments, preferred metabolites of cyclophosphamide for incorporationinto the compositions and formulations disclosed herein include4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard, orcombinations thereof.

In addition, other otic disorders have inflammatory response aspects orare tangentially related to autoimmune conditions, including Meniere'sdisease and non-sudden hearing loss or noise induced hearing loss. Thesedisorders are also explicitly contemplated as benefiting from thecytotoxic agent formulations disclosed herein, and therefore are withinthe scope of the embodiments disclosed.

Meniere's Disease

Meniere's disease is an idiopathic condition characterized by suddenattacks of vertigo, nausea and vomiting that may last for 3 to 24 hours,and may subside gradually. Progressive hearing loss, tinnitus and asensation of pressure in the ears accompanies the disease through time.The cause of Meniere's disease is likely related to an imbalance ofinner ear fluid homeostasis, including an increase in production or adecrease in reabsorption of inner ear fluid.

Studies of the vasopressin (VP)-mediated aquaporin 2 (AQP2) system inthe inner ear suggest a role for VP in inducing endolymph production,thereby increasing pressure in the vestibular and cochlear structures.VP levels were found to be upregulated in endolymphatic hydrops(Meniere's disease) cases, and chronic administration of VP in guineapigs was found to induce endolymphatic hydrops. Treatment with VPantagonists, including infusion of OPC-31260 (a competitive antagonistof V2-R) into the scala tympani resulted in a marked reduction ofMeniere's disease symptoms. Other VP antagonists include WAY-140288,CL-385004, tolvaptan, conivaptan, SR 121463A and VPA 985. (Sanghi et al.Eur. Heart J. (2005) 26:538-543; Palm et al. Nephrol. Dial Transplant(1999) 14:2559-2562).

Other studies suggest a role for estrogen-related receptor β/NR3B2(ERR/Nr3b2) in regulating endolymph production, and therefore pressurein the vestibular/cochlear apparatus. Knock-out studies in micedemonstrate the role of the protein product of the Nr3b2 gene inregulating endolymph fluid production. Nr3b2 expression has beenlocalized in the endolymph-secreting strial marginal cells andvestibular dark cells of the cochlea and vestibular apparatus,respectively. Moreover, conditional knockout of the Nr3b2 gene resultsin deafness and diminished endolymphatic fluid volume. Treatment withantagonists to ERR/Nr3b2 may assist in reducing endolymphatic volume,and thus alter pressure in the auris interna structures.

Other treatments may be aimed at dealing with the immediate symptoms andprevention of recurrence. Low-sodium diets, avoidance of caffeine,alcohol, and tobacco have been advocated. Medications that maytemporarily relieve vertigo attacks include antihistamines (includingmeclizine (Antivert, Bonine, Dramamine, Driminate) and otherantihistamines), and central nervous system agents, includingbarbiturates and/or benzodiazepines, including lorazepam or diazepam.Other examples of drugs that may be useful in relieving symptoms includemuscarinic antagonists, including scopolamine. Nausea and vomiting maybe relieved by suppositories containing antipsychotic agents, includingthe phenothiazine agent prochlorperazine (Compazine, Buccastem, Stemetiland Phenotil).

Surgical procedures that have been used to relieve symptoms include thedestruction of vestibular and/or cochlear function to relieve vertigosymptoms. These procedures aim to either reduce fluid pressure in theinner ear and/or to destroy inner ear balance function. An endolymphaticshunt procedure, which relieves fluid pressure, may be placed in theinner ear to relieve symptoms of vestibular dysfunction. Othertreatments include gentamicin application, which when injected into theeardrum destroys sensory hair cell function, thereby eradicating innerear balance function. Severing of the vestibular nerve may also beemployed, which while preserving hearing, may control vertigo.

Meniere's Syndrome

Meniere's syndrome, which displays similar symptoms as Meniere'sdisease, is attributed as a secondary affliction to another diseaseprocess, e.g. thyroid disease or inner ear inflammation due to syphilisinfection. Meniere's syndrome, thus, are a collection of secondaryeffects to various process that interfere with normal production orresportption of endolymph, including endocrine abnormalities,electrolyte imbalance, autoimmune dysfuntion, medications, infections(e.g. parasitic infections) or hyperlipidemia. Treatment of patientsafflicted with Meniere's syndrome is similar to Meniere's disease.

Vestibular Neuronitis

Vestibular neuronitis is characterized by sudden vertigo attacks, andmay be caused by inflammation of the nerve to the semicircular canalslikely caused by a virus. The first attack of vertigo is usually severe,leading to nystagmus, a condition characterized by the flickering of theeyes involuntarily toward the affected side. Hearing loss does notusually occur. Diagnosis of vestibular neuronitis usually involves testsfor nystagmus using electronystamography, a method of electronicallyrecording eye movements. Magnetic resonance imaging may also beperformed to determine if other causes may play a role in the vertigosymptoms.

Treatment of vertigo is often identical to Meniere's disease, and mayinclude meclizine, lorazepam, prochlorperazine or scopolamine. Fluidsand electrolytes may also be intravenously administered if the vomitingis severe.

Postural Vertigo

Postural vertigo, otherwise known as positional vertigo, ischaracterized by sudden violent vertigo that is triggered by certainhead positions. This condition may be caused by damaged semicircularcanals caused by physical injury to the inner ear, otitis media, earsurgery or blockage of the artery to the inner ear.

Vertigo onset in patients with postural vertigo usually develops when aperson lies on one ear or tilts the head back to look up. Vertigo may beaccompanied by nystagmus. Treatment of postural vertigo often involvesthe same treatment as in Meniere's disease. In severe cases of posturalvertigo, the vestibular nerve is severed to the affected semicircularcanal.

Sensorineural Hearing Loss

Sensorineural hearing loss occurs when the components of the inner earor accompanying neural components are affected, and may contain aneural, i.e. when the auditory nerve or auditory nerve pathways in thebrain are affected, or sensory component. Sensory hearing loss may behereditary, or it may be caused by acoustic trauma (i.e. very loudnoises), a viral infection, drug-induced or Meniere's disease. Neuralhearing loss may occur as a result of brain tumors, infections, orvarious brain and nerve disorders, such as stroke. Some hereditarydiseases, such as Refsum's disease (defective accumulation of branchedfatty acids), may also cause neural disorders affecting hearing loss.Auditory nerve pathways may be damaged by demyelinating diseases, e.g.idiopathic inflammatory demyelinating disease (including multiplesclerosis), transverse myelitis, Devic's disease, progressive multifocalleukoencephalopathy, Guillain-Barre syndrome, chronic inflammatorydemyelinating polyneuropathy and anti-MAG perpheral neuropathy.

The incidence of sudden deafness, or sensorineural hearing loss, occursin about 1 in 5,000 individuals, and may be caused by viral or bacterialinfections, e.g. mumps, measles, influenza, chickenpox, cytomegalovirus,syphillis or infectious mononucleosis, or physical injury to the innerear organ. In some cases, no cause can be identified. In some instances,sensorineural hearing loss is noise induced hearing loss and is causedby acoustic trauma, e.g., loud noise such as gun fire and otherhuman-based noise. Tinnitus and vertigo may accompany sudden deafness,which subsides gradually. Oral corticosteroids are frequently prescribedto treat sensorineural hearing loss. In some cases, surgicalintervention may be necessary.

Hereditary Disorders

Hereditary disorders, including Scheibe, Mondini-Michelle,Waardenburg's, Michel, Alexander's ear deformity, hypertelorism,Jervell-Lange Nielson, Refsum's and Usher's sydromes, are found inapproximately 20% of patients with sensorineural hearing loss.Congenital ear malformations may result from defects in the developmentof the membranous labyrinthine, the osseous labyrinthine, or both. Alongwith profound hearing loss and vestibular function abnormalities,hereditary deformities may also be associated with other dysfunctions,including development of recurring menigitis, cerebral spinal fluid(CSF) leaks, as well as perilymphatic fistulas. Treatment of chronicinfections may be necessitated in hereditary disorder patients.

Cancer of the Ear

Although the cause is unknown, cancer of the ear is often associatedwith long-term and untreated otitis, suggesting a link between chronicinflammation and development of the cancer, at least in some cases.Tumors in the ear can be benign or malignant, and they can exist in theexternal, middle, or inner ear. Symptoms of ear cancer include otorrhea,otalgia, hearing loss, facial palsy, tinnitus, and vertigo. Treatmentoptions are limited, and include surgery, radiotherapy, chemotherapy,and combinations thereof. Also, additional pharmaceutical agents areused to treat symptoms or conditions associated with the cancer,including corticosteroids in the case of facial palsy, and antimicrobialagents when otitis is present.

Systemic administration of conventional cytoxic agents have been used totreat cancer of the ear, including systemic administration ofcyclophosphamide (in CHOP chemotherapy) in combination with radiotherapyand methotrexate, Merkus, P., et al. J. Otorhinolaryngol. Relat. Spec.(2000) 62:274-7, and perfusion of methotrexate through the externalcarotid artery, Mahindrakar, N. H. J. Laryngol. Otol. (1965) 79:921-5.However, treatments requiring systemic administration of the activeagents suffer from the same drawbacks discussed above. Namely,relatively high doses of the agents are required to achieve thenecessary therapeutic doses in the ear, which result in an increase ofundesired, adverse side effects. Accordingly, local administration ofthe cytotoxic agents in the compositions and formulations disclosedherein results in treatment of cancer of the ear with lower effectivedoses, and with a decrease in the incidence and/or severity of sideeffects. Typical side effects of systemic administration of cytotoxicagents, e.g., methotrexate, cyclophosphamide, and thalidomide, for thetreatment of cancer of the ear include anemia, neutropenia, bruising,nausea, dermatitis, hepatitis, pulmonary fibrosis, teratogenicity,peripheral neuropathy, fatigue, constipation, deep vein thrombosis,pulmonary edema, atelectasis, aspiration pneumonia, hypotension, bonemarrow suppression, diarrhea, darkening of skin and nails, alopecia,changes in hair color and texture, lethargy, hemorrhagic cystitis,carcinoma, mouth sores, and decreased immunity.

In certain embodiments, other suitable agents for the treatment ofcancer of the ear that are formulated in the compositions disclosedherein are additional cytotoxic agents, including but not limited toacridine carboxamide, actinomycin,17-N-allylamino-17-demethoxygeldanamycin, amsacrine, aminopterin,anthracycline, antineoplastic, antineoplaston, 5-azacytidine,azathioprine, BL22, bendamustine, biricodar, bleomycin, bortezomib,bryostatin, busulfan, calyculin, camptothecin, capecitabine,carboplatin, chlorambucil, cisplatin, cladribine, clofarabine,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine,dichloroacetic acid, discodermolide, docetaxel, doxorubicin, epirubicin,epothilone, eribulin, estramustine, etoposide, exatecan, exisulind,ferruginol, floxuridine, fludarabine, fluorouracil, fosfestrol,fotemustine, gemcitabine, hydroxyurea, IT-101, idarubicin, ifosfamide,imiquimod, irinotecan, irofulven, ixabepilone, laniquidar, lapatinib,lenalidomide, lomustine, lurtotecan, mafosfamide, masoprocol,mechlorethamine, melphalan, mercaptopurine, mitomycin, mitotane,mitoxantrone, nelarabine, nilotinib, oblimersen, oxaliplatin, PAC-1,paclitaxel, pemetrexed, pentostatin, pipobroman, pixantrone, plicamycin,procarbazine, proteasome inhibitors (e.g., bortezomib), raltitrexed,rebeccamycin, rubitecan, SN-38, salinosporamide A, satraplatin,streptozotocin, swainsonine, tariquidar, taxane, tegafur-uracil,temozolomide, testolactone, thioTEPA, tioguanine, topotecan,trabectedin, tretinoin, triplatin tetranitrate,tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine, vorinostat, and zosuquidar.

Pharmaceutical Agents

Provided herein are cytotoxic agent compositions or formulations thatameliorate or lessen autoimmune otic disorders, including cancer or theear, AIED, and/or inflammatory disorders and their attendant symptoms,which include but are not limited to hearing loss, nystagmus, vertigo,tinnitus, inflammation, swelling, infection and congestion. Oticdisorders, including AIED and/or inflammatory disorders, have causes andsymptoms that are responsive to the pharmaceutical agents disclosedherein, or other pharmaceutical agents. Also provided herein arecytotoxic agent compositions and formulations that treat, ameliorate, orlessen cancer, particularly cancer of the ear. Cytotoxic agents that arenot disclosed herein but which are useful for the amelioration oreradication of otic disorders are expressly included and intended withinthe scope of the embodiments presented.

Moreover, pharmaceutical agents which have been previously shown to beexcessively toxic, harmful or non-effective during systemic or localizedapplication in other organ systems, for example through toxicmetabolites formed after hepatic processing, toxicity of the drug inparticular organs, tissues or systems, through high levels needed toachieve efficacy, through the inability to be released through systemicpathways, or through poor PK characteristics, are useful in someembodiments herein. Accordingly, pharmaceutical agents which havelimited or no systemic release, systemic toxicity, poor PKcharacteristics or combinations thereof are contemplated within thescope of the embodiments disclosed herein.

The cytotoxic agent formulations disclosed herein are optionallytargeted directly to otic structures where treatment is needed; forexample, one embodiment contemplated is the direct application of thecytotoxic agent formulations disclosed herein onto the round windowmembrane or the crista fenestrae cochlea of the auris interna, allowingdirect access and treatment of the auris interna, or inner earcomponents. In other embodiments, the cytotoxic agent formulationsdisclosed herein are applied directly to the oval window. In yet otherembodiments, direct access is obtained through microinjection directlyinto the auris interna, for example, through cochlear microperfusion.Such embodiments also optionally comprise a drug delivery device,wherein the drug delivery device delivers the cytotoxic agentformulations through use of a needle and syringe, a pump, amicroinjection device or any combination thereof. In still otherembodiments, application of the cytotoxic agent formulation is targetedto the auris media through piercing of the intratympanic membrane andapplying the cytotoxic agent formulation directly to the auris mediastructures affected, including the walls of the tympanic cavity orauditory ossicles. By doing so, the cytotoxic agent formulationsdisclosed herein are confined to the targeted auris media structure, andwill not be lost, for example, through diffusion or leakage through theeustachian tube or pierced tympanic membrane. In some embodiments,cytotoxic agent formulations and compositions are delivered to the aurisexterna in any suitable manner, including by ear wick, cotton swab, orear drops. Also, in other embodiments, the cytotoxic agent formulationsare targeted to specific regions of the auris externa by applicationwith a needle and syringe, a pump, a microinjection device, or anycombination thereof. For example, in the case of treatment of earcancer, cytotoxic agent formulations disclosed herein are delivereddirectly to tumors and lesions by way of any suitable means, includingwith a needle and syringe, a pump, a microinjection device, or anycombination thereof.

Some pharmaceutical agents, either alone or in combination, areototoxic. For example, some chemotherapeutic agents, includingactinomycin, bleomycin, cisplatin, carboplatin and vincristine; andantibiotics, including erythromycin, gentamicin, streptomycin,dihydrostreptomycin, tobramycin, netilmicin, amikacin, neomycin,kanamycin, etiomycin, vancomycin, metronidizole, capreomycin, are mildlyto very ototoxic, and affect the vestibular and cochlear structuresdifferentially. However, in some instances, the combination of anototoxic drug, for example cisplatin, in combination with anotoprotectant lessens the ototoxic effects of the drug. Moreover, thelocalized application of the potentially ototoxic drug also lessens thetoxic effects that otherwise occur through systemic application throughthe use of lower amounts with maintained efficacy, and/or the use oftargeted amounts for a shorter period of time.

Some pharmaceutical excipients, diluents or carriers are alsopotentially ototoxic. For example, benzalkonium chloride, a commonpreservative, is ototoxic at certain levels and therefore potentiallyharmful if introduced into the vestibular or cochlear structures. Informulating a controlled release cytotoxic agent formulation, it isadvised to avoid or combine the appropriate excipients, diluents orcarriers to lessen or eliminate potential ototoxic components from theformulation, or to decrease the amount of such excipients, diluents orcarriers. The ototoxicity of the pharmaceutical agents, excipients,diluents, carriers, or formulations and compositions disclosed hereincan be ascertained using an accepted animal model. See, e.g., Maritini,A., et al. Ann. N.Y. Acad. Sci. (1999) 884:85-98. In some embodiments, acontrolled release cytotoxic agent formulation disclosed hereinoptionally includes otoprotective agents, such as antioxidants, alphalipoic acid, calicum, fosfomycin or iron chelators, to counteractpotential ototoxic effects that may arise from the use of specifictherapeutic agents or excipients, diluents or carriers.

Cytotoxic Agents

Any cytotoxic agent useful for the treatment of otic disorders, e.g.,inflammatory diseases of the ear or cancer of the ear, is suitable foruse in the formulations and methods disclosed herein. In certainembodiments, the cytotoxic agent is an antimetabolite, an antifolate, analkylating agentand/or a DNA intercalator. In some embodiments, thecytotoxic agent is a protein, a peptide, an antibody, DNA, acarbohydrate, an inorganic molecule, or an organic molecule. In certainembodiments, the cytotoxic agents are cytotoxic small molecules.Typically, cytotoxic small molecules are of relatively low molecularweight, e.g., less than 1,000, or less than 600-700, or between 300-700molecular weight. In some embodiments, the cytotoxic small moleculeswill also have anti-inflammatory properties.

In certain embodiments, the cytotoxic agents are methotrexate(RHEUMATREX®, Amethopterin) cyclophosphamide (CYTOXAN®), and thalidomide(THALIDOMID®). All of the compounds can be used to treat cancer,including cancer of the ear. Further, all of the compounds haveanti-inflammatory properties and can be used in the formulations andcompositions disclosed herein for the treatment of inflammatorydisorders of the ear, including AIED.

Although systemic administration of methotrexate, cyclophosphamide, andthalidomide is currently used to treat or is being investigated for thetreatment of otic disorders, such as inflammatory otic disorders,including AIED, Meniere's disease, and Behçet's disease, as well ascancer of the ear, the cytotoxic agents are not without the potentialfor serious adverse side effects. Moreover, cytotoxic agents whichdemonstrate efficacy but are otherwise not approvable because of safetyconsiderations is also contemplated within the embodiments disclosedherein. It is contemplated that localized application of the cytotoxicagents to the target otic structures for treatment of autoimmune and/orinflammatory disorders, as well as cancer of the ear, will result in thereduction or elimination of adverse side effects experienced withsystemic treatment. Moreover, localized treatment with the cytotoxicagents contemplated herein will also reduce the amount of agent neededfor effective treatment of the targeted disorder due, for example, toincreased retention of the active agents in the auris interna and/ormedia, to the existence of the biological blood barrier in the aurisinterna, or to the lack of sufficient systemic access to the aurismedia.

In some embodiments, cytotoxic agents used in the compositions,formulations, and methods disclosed herein are metabolites, salts,polymorphs, prodrugs, analogues, and derivatives of cytotoxic agents,including methotrexate, cyclophosphamide, and thalidomide. Particularlypreferred are metabolites, salts, polymorphs, prodrugs, analogues, andderivatives of cytotoxic agents, e.g., methotrexate, cyclophosphamide,and thalidomide, that retain at least partially the cytotoxicity andanti-inflammatory properties of the parent compounds. In certainembodiments, analogues of thalidomide used in the formulations andcompositions disclosed herein are lenalidomide (REVLIMID®) and CC-4047(ACTIMID®).

Cyclophosphamide is a prodrug that undergoes in vivo metabolism whenadministered systemically. The oxidized metabolite4-hydroxycyclophosphamide exists in equilibrium with aldophosphamide,and the two compounds serve as the transport forms of the active agentphosphoramide mustard and the degradation byproduct acrolein. Thus, insome embodiments, preferred cyclophosphamide metabolites forincorporation into the formulations and compositions disclosed hereinare 4-hydroxycyclophosphamide, aldophosphamide, phosphoramide mustard,and combinations thereof.

Other cytotoxic agents used in the compositions, formulations, andmethods disclosed herein, particularly for the treatment of cancer ofthe ear, are any conventional chemotherpeutic agents, including acridinecarboxamide, actinomycin, 17-N-allylamino-17-demethoxygeldanamycin,aminopterin, amsacrine, anthracycline, antineoplastic, antineoplaston,5-azacytidine, azathioprine, BL22, bendamustine, biricodar, bleomycin,bortezomib, bryostatin, busulfan, calyculin, camptothecin, capecitabine,carboplatin, chlorambucil, cisplatin, cladribine, clofarabine,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine,dichloroacetic acid, discodermolide, docetaxel, doxorubicin, epirubicin,epothilone, eribulin, estramustine, etoposide, exatecan, exisulind,ferruginol, floxuridine, fludarabine, fluorouracil, fosfestrol,fotemustine, gemcitabine, hydroxyurea, IT-101, idarubicin, ifosfamide,imiquimod, irinotecan, irofulven, ixabepilone, laniquidar, lapatinib,lenalidomide, lomustine, lurtotecan, mafosfamide, masoprocol,mechlorethamine, melphalan, mercaptopurine, mitomycin, mitotane,mitoxantrone, nelarabine, nilotinib, oblimersen, oxaliplatin, PAC-1,paclitaxel, pemetrexed, pentostatin, pipobroman, pixantrone, plicamycin,procarbazine, proteasome inhibitors (e.g., bortezomib), raltitrexed,rebeccamycin, rubitecan, SN-38, salinosporamide A, satraplatin,streptozotocin, swainsonine, tariquidar, taxane, tegafur-uracil,temozolomide, testolactone, thioTEPA, tioguanine, topotecan,trabectedin, tretinoin, triplatin tetranitrate,tris(2-chloroethyl)amine, troxacitabine, uracil mustard, valrubicin,vinblastine, vincristine, vinorelbine, vorinostat, and zosuquidar.

Immune System Cells

In some embodiments, local otic therapy comprises the use of cells whichparticipate in induction of cytotoxicity. In some embodiments, the cellswhich participate in induction of cytotoxicity are T cells. Cytotoxic Tcells are a sub-group of T lymphocytes (a type of white blood cell) thatkill cells that are infected or are otherwise damaged or dysfunctional.In some instances, administration of proteins (e.g., cytokines such asIL-2) results in recruitment and/or differentiation of cytotoxic T cellsin auris structures (e.g., in the cochlear region). Also contemplatedwithin the scope of embodiments described herein is the administrationof small molecule agents that induce recruitment and/or differentiationof cytotoxic T cells in auris structures (e.g., in the cochlear region).In some embodiments, administration of epitopes and/or antigens (e.g.,fragments of viral proteins) results in recruitment and/ordifferentiation of cytotoxic T cells in auris structures (e.g., in thecochlear region).

Combination Therapy Anti-TNF Agents

Contemplated for use in conjunction with the cytotoxic agentformulations disclosed herein are agents that reduce or amelioratesymptoms or effects resulting from an autoimmune disease and/orinflammatory disorder, including AIED or OM. Accordingly, someembodiments incorporate the use of agents which block the effects ofTNF-α, including anti-TNF agents. By way of example only, anti-TNFagents include etanercept (ENBREL®), infliximab (REMICADE®), adalimumab(HUMIRA®), and golimumab (CNTO148) or combinations thereof.

Ifliximab and adalimumab are anti-TNF monoclonal antibodies, andetanercept is a fusion protein designed to bind specifically to the TNFprotein. All are currently approved for use in the treatment ofrheumatoid arthritis. Golimumab, which is currently in Phase 3 clinicaltrials for rheumatoid arthritis, psoriatic arthritis and ankylosingspondylitis, is a fully-humanized anti-TNF-α IgG1 monoclonal antibodythat targets and neutralizes both the soluble and the membrane-boundform of TNF-α.

Other antagonists of TNF, by way of example only, include TNF receptors(pegylated soluble TNF receptor type 1; Amgen); TNF binding factors(Onercept; Serono); TNF antibodies (US Patent App. No. 2005/0123541; USPatent App. No. 2004/0185047); single domain antibodies against the p55TNF receptor (US Patent App. No. 2008/00088713); soluble TNF receptors(US Patent App. No. 2007/0249538); fusion polypeptides binding to TNF(US Patent App. No. 2007/0128177); TNF-α converting enzyme inhibitors(Skotnicki et al., Annual Reports in Medicinal Chemistry (2003), 38,153-162); IKK inhibitors (Karin et al., Nature Reviews Drug Discovery(2004), 3, 17-26) and flavone derivatives (US Patent App. No.2006/0105967), all of which are incorporated by reference for suchdisclosure.

The use of Onercept, a soluble TNF p55 receptor, was discontinued in2005. Three phase-III clinical trials reported patients diagnosed withfatal sepsis. A risk to benefit analysis was subsequently performed,resulting in the discontinuation of the clinical trials. As discussedabove, the embodiments herein specifically encompass the use of anti-TNFagents that have been previously shown to have limited or no systemicrelease, systemic toxicity, poor PK characteristics of combinationsthereof.

Anti-Emetic Agents/Central Nervous System Agents

Anti-emetic agents are optionally used in combination with the cytotoxicagent formulations disclosed herein. Anti-emetic agents includeantihistamines and central nervous agents, including anti-psychoticagents, barbiturates, benzodiazepines and phenothiazines. Otheranti-emetic agents include the serotonin receptor antagonists, whichinclude dolasetron, granisetron, ondansetron, tropisetron, palonosetron,and combinations thereof; dopamine antagonists, including domperidone,properidol, haloperidol, chlorpromazine, promethazine, prochlorperazineand combinations thereof; cannabinoids, including dronabinol, nabilone,sativex, and combinations thereof; anticholinergics, includingscopolamine; and steroids, including dexamethasone; trimethobenzamine,emetrol, propofol, muscimol, and combinations thereof.

Optionally, central nervous system agents and barbiturates are useful inthe treatment of nausea and vomiting, symptoms that often accompany oticdisorders. When used, an appropriate barbiturate and/or central nervoussystem agent is selected to relieve or ameliorate specific symptomswithout possible side effects, including ototoxicity. Moreover, asdiscussed above, targeting of the drugs to the round window membrane ofthe auris interna reduces possible side effects and toxicity caused bysystemic administration of these drugs. Barbiturates, which act as acentral nervous system depressant, include allobarbital, alphenal,amobarbital, aprobarbital, barnexaclone, barbital, brallobarbital,butabarbital, butalbital, butallylonal, butobarbital, corvalol,crotylbarbital, cyclobarbital, cyclopal, ethallobarbital, febarbamate,heptabarbital, hexethal, hexobarbital, metharbital, methohexital,methylphenobarbital, narcobarbital, nealbarbital, pentobarbital,phenobarbital, primidone, probarbital, propallylonal, proxibarbital,reposal, secobarbital, sigmodal, sodium thiopental, talbutal,thialbarbital, thiamylal, thiobarbital, thiobutabarbital, tuinal,valofane, vinbarbital, vinylbital, and combinations thereof.

Other central nervous system agents which are optionally used inconjunction with the cytotoxic agent formulations disclosed hereininclude benzodiazepines or phenothiazines. Useful benzodiazepinesinclude, but are not limited to diazepam, lorazepam, oxazepam, prazepam,alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate,brotizolam, estazolam, flunitrazepam, flurazepam, loprazolam,lormetazepam, midazolam, nimetazepam, nitrazepam, ternazepam, triazolam,and combinations thereof. Examples of phenothiazines includeprochlorperazine, chlorpromazine, promazine, triflupromazine,levopromazine, methotrimepramazine, mesoridazine, thiroridazine,fluphenazine, perphenazine, flupentixol, trifluoperazine, andcombinations thereof.

Antihistamines, or histamine antagonists, act to inhibit the release oraction of histamine. Antihistamines that target the H1 receptor areuseful in the alleviation or reduction of nausea and vomiting symptomsthat are associated with AIED, other autoimmune disorders, as well asanti-inflammatory disorders. Such antihistamines include, but are notlimited to, meclizine, diphenhydramine, loratadine and quetiapine. Otherantihistamines include mepyramine, piperoxan, antazoline, carbinoxamine,doxylamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine,chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine,cyclizine, chlorcyclizine, hydroxyzine, promethazine, alimemazine,trimeprazine, cyproheptadine, azatadine, ketotifen, oxatomide andcombinations thereof.

Antimicrobial Agents

Antimicrobial agents are also contemplated as useful with theformulations disclosed herein. Antimicrobial agents include agents thatact to inhibit or eradicate microbes, including bacteria, fungi orparasites. Specific antimicrobial agents may be used to combat specificmicrobes. Accordingly, a skilled practitioner would know whichantimicrobial agent would be relevant or useful depending on the microbeidentified, or the symptoms displayed. Antimicrobial agents includeantibiotics, antiviral agents, antifungal agents, and antiparasiticagents.

Antibiotics may include amikacin, gentamicin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, paromomycin, geldanmycin,herbimycin, loracarbef, ertapenem, doripenem, imipenem, cilastatin,meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor,cefamandole, cefoxitin, defprozil, cefuroxime, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole,teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin,spectinomycin, aztreonam, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillan,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovfloxacin, mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanimilimde, sulfsalazine, sulfsioxazole,trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline,tetracycline, arsphenamine, chloramphenicol, clindamycin, lincomycin,ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,pyrazinamide, quinuspristin/dalfopristin, rifampin, timidazole, andcombinations thereof.

Antiviral agents may include acyclovir, famciclovir and valacyclovir.Other antiviral agents include abacavir, aciclovir, adfovir, amantadine,amprenavir, arbidol, atazanavir, artipla, brivudine, cidofovir,combivir, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,fomvirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, gardasil,ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine,integrase inhibitors, interferons, including interferon type III,interferon type II, interferon type I, lamivudine, lopinavir, loviride,MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir,nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril,podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors,ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir,tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine,truvada, valganciclovir, vicriviroc, vidarabine, viramidine,zalcitabine, zanamivir, zidovudine, and combinations thereof.

Antifungal agents may include ammolfine, utenafine, naftifine,terbinafine, flucytosine, fluconazole, itraconazole, ketoconazole,posaconazole, ravuconazole, voriconazole, clotrimazole, econazole,miconazole, oxiconazole, sulconazole, terconazole, tioconazole,nikkomycin Z, caspofungin, micafungin, anidulafungin, amphotericin B,liposomal nystastin, pimaricin, griseofulvin, ciclopirox olamine,haloprogin, tolnaftate, undecylenate, and combinations thereof.Antiparasitic agents may include amitraz, amoscanate, avermectin,carbadox, diethylcarbamizine, dimetridazole, diminazene, ivermectin,macrofilaricide, malathion, mitaban, oxamniquine, permethrin,praziquantel, prantel pamoate, selamectin, sodium stibogluconate,thiabendazole, and combinations thereof.

Platelet Activating Factor Antagonists

Platelet activating factor antagonists are also contemplated for use incombination with the cytotoxic agent formulations disclosed herein.Platelet activating factor antagonists include, by way of example only,kadsurenone, phomactin G, ginsenosides, apafant(4-(2-chlorophenyl)-9-methyl-2[3(4-morpholinyl)-3-propanol-1-yl[6H-thieno[3.2f-[[1,2,4]triazolo]4,3-1]]1.4]diazepine),A-85783, BN-52063, BN-52021, BN-50730 (tetrahedra-4,7,8,10 methyl-1(chloro-1 phenyl)-6 (methoxy-4 phenyl-carbamoyl)-9 pyrido[4′,3′-4,5]thieno[3,2-f]triazolo-1,2,4 [4,3-a] diazepine-1,4), BN 50739, SM-12502,RP-55778, Ro 24-4736, SR27417A, CV-6209, WEB 2086, WEB 2170,14-deoxyandrographolide, CL 184005, CV-3988, TCV-309, PMS-601, TCV-309and combinations thereof.

Nitric Oxide Synthase Inhibitors

Nitric oxide synthase (NOS) inhibitors are also contemplated for use incombination with the cytotoxic agent formulations disclosed herein. NOSinhibitors include, by way of example only, aminoguanidine,1-Amino-2-hydroxyguanidine p-Toluensulfate, guanidinoethyldisulfide(GED), Bromocriptine Mesylate, Dexamethasone, NG,NG-Dimethyl-L-arginine,Dihydrochloride, Diphenyleneiodonium Chloride, 2-Ethyl-2-thiopseudourea,haloperidol, L-N5-(1-Iminoethyl)ornithine, MEG, S-MethylisothioureaSulfate (SMT), S-Methyl-L-thiocitrulline, NG-Monoethyl-L-arginine,NG-Monomethyl-D-arginine, NG-Nitro-L-arginine Methyl Ester, L-NIL,NG-Nitro-L-arginine (L-NNA), 7-Nitroindazole, nNOS Inhibitor I,1,3-PBITU, L-Thiocitrulline, NG-Propyl-L-arginine, SKF-525A, TRIM,NG-nitro-L-arginine methyl ester (L-NAME), MTR-105, L-NMMA, BBS-2,ONO-1714 and combinations thereof.

Otoprotectants

In some embodiments, any otic formulation described herein furthercomprises otoprotectants that reduce, inhibit or ameliorate theototoxicity of cytotoxic agents such as cytotoxic agents or antibioticsas described herein. Examples of otoprotectants include, and are notlimited to, thiols and/or thiol derivatives and/or pharmaceuticallyacceptable salts, or derivatives (e.g. prodrugs) thereof (e.g.,D-methionine, L-methionine, ethionine, hydroxyl methionine, methioninol,amifostine, mesna (sodium 2-sulfanylethanesulfonate), a mixture of D andL methionine, normethionine, homomethionine, S-adenosyl-L-methionine),diethyldithiocarbamate, ebselen(2-phenyl-1,2-benzisoselenazol-3(2H)-one), sodium thiosulfate, AM-111 (acell permeable JNK inhibitor, (Laboratoires Auris SAS)), leucovorin,leucovorin calcium, dexrazoxane, piracetam, Oxiracetam, Aniracetam,Pramiracetam, Phenylpiracetam (Carphedon), Etiracetam, Levetiracetam,Nefiracetam, Nicoracetam, Rolziracetam, Nebracetam, Fasoracetam,Coluracetam, Dimiracetam, Brivaracetam, Seletracetam, Rolipramand orcombinations thereof. Otoprotectants allow for the administration ofotic agents (e.g., cytotoxic agents described herein) at doses that arehigher than conventional doses; the otic agents would otherwise besystemically administered at lower doses because of associatedototoxicity.

Other Agents

Other pharmaceutical agents that are optionally used in combination withthe cytotoxic agent formulations disclosed herein for the treatment ofotic disorders, e.g., inflammatory disorders of the ear and/or cancer ofthe ear, include other agents that have been used to treat inflammatorydisorders and/or cancer of the ear, such as corticosteroids;chemotherapeutic agents, including additional chemotherapeutic agentsthat are not cytotoxic (e.g., agents that are given to treat symptoms ofcancer or side effects of other chemotherapeutic agents), treatment withcollagen, gamma globulin, interferons, and/or copaxone; and combinationsthereof. In addition, other pharmaceutical agents are optionally used totreat attendant symptoms of AIED, other autoimmune and/or inflammatorydisorders of the ear, and cancer of the ear, including vomiting,dizziness and general malaise. The additional active agents can beformulated with the cytotoxic agents in the compositions andformulations disclosed herein, or they can be administered separatelythrough alternative modes of delivery. Any combination of one or morepharmaceutical agents (e.g., cytotoxic agents) and one or moreadditional therapeutic agents is compatible with the compositions anddevices described herein.

Otic Surgery and Implants

In some embodiments, the pharmaceutical formulations, compositions ordevices described herein are used in combination with implants (e.g.,implantation, short-term use, long-term use, or removal of). As usedherein, implants include auris-interna or auris-media medical devices,examples of which include cochlear implants, hearing sparing devices,hearing-improvement devices, short electrodes, micro-prostheses orpiston-like prostheses; needles; stem cell transplants; drug deliverydevices; any cell-based therapeutic; or the like. In some instances, theimplants are used in conjunction with a patient experiencing hearingloss. In some instances, the hearing loss is present at birth. In someinstances, the hearing loss is associated with conditions such ascancer, AIED, bacterial meningitis or the like that lead toosteoneogenesis and/or nerve damage with rapid obliteration of cochlearstructures and profound hearing loss.

In some instances, an implant is a stem cell transplant in the ear. Insome instances, an implant is a small electronic device that has anexternal portion placed behind the ear, and a second portion that issurgically placed under the skin that helps provide a sense of sound toa person who is profoundly deaf or severely hard-of-hearing. By way ofexample, such cochlear medical device implants bypass damaged portionsof the ear and directly stimulate the auditory nerve. In some instancescochlear implants are used in single sided deafness. In some instancescochlear implants are used for deafness in both ears.

In some embodiments, administration of a cytotoxic agent composition ordevice described herein in combination with a medical device implant ora cell-based transplant delays or prevents collateral damage to aurisstructures, e.g., irritation, cell death, osteoneogeneis, caused byinstallation of an external device and/or cells in the ear. In someembodiments, administration of a cytotoxic agent composition or devicedescribed herein in combination with an implant allows for a moreeffective restoration of hearing loss compared to an implant alone.

In some embodiments, administration of a cytotoxic agent composition ordevice described herein reduces damage to cochlear structures caused byunderlying conditions (e.g., bacterial meningitis, cancer, autoimmuneear disease (AIED)) allowing for successful cochlear deviceimplantation. In some embodiments, administration of a composition ordevice described herein, in conjunction with otic surgery, medicaldevice implantation and/or cell transplantation, reduces or preventscell death (e.g., cell death in auris interna or auris media) associatedwith otic surgery, medical device implantation and/or celltransplantation.

In some embodiments, administration of a cytotoxic agent composition ordevice described herein (e.g., a composition or device comprisingcytotoxic agent and an antimicrobial) in conjunction with a cochlearimplant or stem cell transplant has a trophic effect (e.g., promoteshealthy growth of cells and/or healing of tissue in the area of animplant or transplant). In some embodiments, a trophic effect isdesirable during otic surgery or during intratympanic injectionprocedures. In some embodiments, a trophic effect is desirable afterinstallation of a medical device or after a cell transplant. In some ofsuch embodiments, the cytotoxic agent compositions or devices describedherein are administered via direct cochlear injection, through achochleostomy or via deposition on the round window.

In some embodiments, administration of an anti-inflammatory composition(e.g., a composition comprising an anti-TNF agent or an antimicrobialagent in combination with a cytotoxic agent) reduces inflammation and/orinfections associated with otic surgery, implantation of a medicaldevice or a cell transplant. In some instances, perfusion of a surgicalarea with a cytotoxic agent formulation described herein reduces oreliminates post-surgical and/or post-implantation complications (e.g.,inflammation, osteoneogenesis or the like). In some instances, perfusionof a surgical area with a formulation described herein reducespost-surgery or post-implantation recuperation time.

In one aspect, the formulations described herein, and modes ofadministration thereof, are applicable to methods of direct perfusion ofthe inner ear compartments. Thus, the formulations described herein areuseful in combination with surgical procedures including, by way ofnon-limiting examples, cochleostomy, labyrinthotomy, mastoidectomy,stapedectomy, stapedotomy, endolymphatic sacculotomy or the like. Insome embodiments, the inner ear compartments are perfused with aformulation described herein prior to otic surgery, during otic surgery,after otic surgery, or a combination thereof. In some of suchembodiments, the formulations described herein are substantially free ofextended release components (e.g., gelling components such aspolyoxyethylene-polyoxypropylene copolymers). In some of suchembodiments, the formulations described herein contain less than 5% ofthe extended release components (e.g., gelling components such aspolyoxyethylene-polyoxypropylene triblock copolymers) by weight of theformulation. In some of such embodiments, the formulations describedherein contain less than 2% of the extended release components (e.g.,gelling components such as polyoxyethylene-polyoxypropylene triblockcopolymers) by weight of the formulation. In some of such embodiments,the formulations described herein contain less than 1% of the extendedrelease components (e.g., gelling components such aspolyoxyethylene-polyoxypropylene triblock copolymers) by weight of theformulation. In some of such embodiments, a composition described hereinthat is used for perfusion of a surgical area contains substantially nogelling component.

In some embodiments, the compositions described herein have aconcentration of active pharmaceutical ingredient between about 0.01% toabout 90%, between about 0.01% to about 50%, between about 0.1% to about70%, between about 0.1% to about 50%, between about 0.1% to about 40%,between about 0.1% to about 30%, between about 0.1% to about 20%,between about 0.1% to about 10%, or between about 0.1% to about 5%, ofthe active ingredient, or pharmaceutically acceptable prodrug or saltthereof, by weight of the composition. In some embodiments, thecompositions described herein have a concentration of activepharmaceutical agent between about 1% to about 50%, between about 5% toabout 50%, between about 10% to about 40%, or between about 10% to about30%, of the active ingredient, or pharmaceutically acceptable prodrug orsalt thereof, by weight of the composition. In some embodiments,formulations described herein comprise about 70% by weight of acytotoxic agent by weight of the formulation. In some embodiments,formulations described herein comprise about 60% by weight of acytotoxic agent by weight of the formulation. In some embodiments,formulations described herein comprise about 50% by weight of acytotoxic agent by weight of the formulation. In some embodiments,formulations described herein comprise about 40% by weight of acytotoxic agent by weight of the formulation. In some embodiments,formulations described herein comprise about 30% by weight of acytotoxic agent by weight of the formulation. In some embodiments,formulations described herein comprise about 20% by weight of acytotoxic agent by weight of the formulation. In some embodiments,formulations described herein comprise about 15% by weight of acytotoxic agent, or pharmaceutically acceptable prodrug or salt thereof,by weight of the formulation. In some embodiments, formulationsdescribed herein comprise about 10% by weight of a cytotoxic agent byweight of the formulation. In some embodiments, formulations describedherein comprise about 5% by weight of a cytotoxic agent, orpharmaceutically acceptable prodrug or salt thereof, by weight of theformulation. In some embodiments, formulations described herein compriseabout 2.5% by weight of a cytotoxic agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, formulations described herein comprise about 1% byweight of a cytotoxic agent, or pharmaceutically acceptable prodrug orsalt thereof, by weight of the formulation. In some embodiments,formulations described herein comprise about 0.5% by weight of acytotoxic agent, or pharmaceutically acceptable prodrug or salt thereof,by weight of the formulation. In some embodiments, formulationsdescribed herein comprise about 0.1% by weight of a cytotoxic agent, orpharmaceutically acceptable prodrug or salt thereof, by weight of theformulation. In some embodiments, formulations described herein compriseabout 0.01% by weight of a cytotoxic agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, the formulations described herein have a concentrationof active pharmaceutical ingredient, or pharmaceutically acceptableprodrug or salt thereof, between about 0.1 to about 70 mg/mL, betweenabout 0.5 mg/mL to about 70 mg/mL, between about 0.5 mg/mL to about 50mg/mL, between about 0.5 mg/mL to about 20 mg/mL, between about 1 mg toabout 70 mg/mL, between about 1 mg to about 50 mg/mL, between about 1mg/mL and about 20 mg/mL, between about 1 mg/mL to about 10 mg/mL, orbetween about 1 mg/mL to about 5 mg/mL, of the active agent, orpharmaceutically acceptable prodrug or salt thereof, by volume of theformulation.

Presented below (Table 1) are examples of active agents contemplated foruse with the formulations and devices disclosed herein. One or moreactive agents are used in any of the formulations or devices describedherein.

Active Agents (including pharmaceutically acceptable salts of theseactive agents) for use with the Formulations Disclosed Herein

TABLE 1 Auris Condition Therapeutic Agent Benign ParoxysmalDiphenhydramine Positional Vertigo Benign Paroxysmal LorazepamPositional Vertigo Benign Paroxysmal Meclizine Positional Vertigo BenignParoxysmal Oldansetron Positional Vertigo Hearing Loss Estrogen AIEDCyclophosphamide AIED cisplatin Cancer Cyclohosphamide Hearing LossEstrogen and progesterone (E + P) Hearing Loss Folic acid Hearing LossLactated Ringer's with 0.03% Ofloxacin Hearing Loss Methotrexate HearingLoss N-acetyl cysteine Meniere's Disease Betahistine Meniere's DiseaseSildenafil Meniere's Disease conivaptan Middle Ear EffusionPneumonococcal vaccine Otitis Externa Diclofenac sodium; dexotc OtitisExterna, Acute AL-15469A/AL-38905 Otitis Media Amoxicillin/clavulanateOtitis Media Dornase alfa Otitis Media Echinacea purpurea Otitis MediaFaropenem medoxomil Otitis Media Levofloxacin Otitis Media PNCRM9 OtitisMedia Pneumococcal vaccine Otitis Media Telithromycin Otitis Media ZmaxOtitis Media with Lansoprazole Effusion Otitis Media, Acute AL-15469A;AL-38905 Otitis Media, Acute Amoxicillin Otitis Media, AcuteAmoxicillin-clavulanate Otitis Media, Acute Azithromycin Otitis Media,Acute Azithromycin SR Otitis Media, Acute Cefdinir Otitis Media, AcuteHyland's earache drops Otitis Media, Acute Montelukast Otitis Media,Acute Pneumonococcal vaccine Otitis Media, Acute AL-15469A/AL38905 withTypanostomy Tubes Otitis Media, Chronic Sulfamethoxazole- trimethoprimOtitis Media, Azithromycin Suppurative Otitis Media, TelithromycinSuppurative Otosclerosis Acetylcysteine Ototoxicity Aspirin TinnitusAcamprosate Tinnitus Gabapentin Tinnitus Modafinil Tinnitus NeramexaneTinnitus Neramexane mesylate Tinnitus Piribedil Tinnitus VardenafilTinnitus Vestipitant + Paroxetine Tinnitus Vestiplitant Tinnitus Zincsulfate

General Methods of Sterilization

Provided herein are otic compositions that ameliorate or lessen oticdisorders described herein. Further provided herein are methodscomprising the administration of said otic compositions. In someembodiments, the compositions or devices are sterilized. Included withinthe embodiments disclosed herein are means and processes forsterilization of a pharmaceutical composition or device disclosed hereinfor use in humans. The goal is to provide a safe pharmaceutical product,relatively free of infection causing micro-organisms. The U.S. Food andDrug Administration has provided regulatory guidance in the publication“Guidance for Industry: Sterile Drug Products Produced by AsepticProcessing” available at: http://www.fda.gov/cder/guidance/5882fnl.htm,which is incorporated herein by reference in its entirety.

As used herein, sterilization means a process used to destroy or removemicroorganisms that are present in a product or packaging. Any suitablemethod available for sterilization of objects and compositions is used.Available methods for the inactivation of microorganisms include, butare not limited to, the application of extreme heat, lethal chemicals,or gamma radiation. In some embodiments is a process for the preparationof an otic therapeutic formulation comprising subjecting the formulationto a sterilization method selected from heat sterilization, chemicalsterilization, radiation sterilization or filtration sterilization. Themethod used depends largely upon the nature of the device or compositionto be sterilized. Detailed descriptions of many methods of sterilizationare given in Chapter 40 of Remington: The Science and Practice ofPharmacy published by Lippincott, Williams & Wilkins, and isincorporated by reference with respect to this subject matter.

Sterilization by Heat

Many methods are available for sterilization by the application ofextreme heat. One method is through the use of a saturated steamautoclave. In this method, saturated steam at a temperature of at least121° C. is allowed to contact the object to be sterilized. The transferof heat is either directly to the microorganism, in the case of anobject to be sterilized, or indirectly to the microorganism by heatingthe bulk of an aqueous solution to be sterilized. This method is widelypracticed as it allows flexibility, safety and economy in thesterilization process.

Dry heat sterilization is a method which is used to kill microorganismsand perform depyrogenation at elevated temperatures. This process takesplace in an apparatus suitable for heating HEPA-filteredmicroorganism-free air to temperatures of at least 130-180° C. for thesterilization process and to temperatures of at least 230-250° C. forthe depyrogenation process. Water to reconstitute concentrated orpowdered formulations is also sterilized by autoclave. In someembodiments, the formulations described herein comprise micoronizedcytotoxic agents (e.g., micro-thalidomide powder) that are sterilized bydry heating, e.g., heating for about 7-11 hours at internal powdertemperatures of 130-140° C., or for 1-2 hours at internal temperaturesof 150-180° C.

Chemical Sterilization

Chemical sterilization methods are an alternative for products that donot withstand the extremes of heat sterilization. In this method, avariety of gases and vapors with germicidal properties, such as ethyleneoxide, chlorine dioxide, formaldehyde or ozone are used as theanti-apoptotic agents. The germicidal activity of ethylene oxide, forexample, arises from its ability to serve as a reactive alkylatingagent. Thus, the sterilization process requires the ethylene oxidevapors to make direct contact with the product to be sterilized.

Radiation Sterilization

One advantage of radiation sterilization is the ability to sterilizemany types of products without heat degradation or other damage. Theradiation commonly employed is beta radiation or alternatively, gammaradiation from a ⁶⁰Co source. The penetrating ability of gamma radiationallows its use in the sterilization of many product types, includingsolutions, compositions and heterogeneous mixtures. The germicidaleffects of irradiation arise from the interaction of gamma radiationwith biological macromolecules. This interaction generates chargedspecies and free radicals. Subsequent chemical reactions, such asrearrangements and cross-linking processes, result in the loss of normalfunction for these biological macromolecules. The formulations describedherein are also optionally sterilized using beta irradiation.

Filtration

Filtration sterilization is a method used to remove but not destroymicroorganisms from solutions. Membrane filters are used to filterheat-sensitive solutions. Such filters are thin, strong, homogenouspolymers of mixed cellulosic esters (MCE), polyvinylidene fluoride (PVF;also known as PVDF), or polytetrafluoroethylene (PTFE) and have poresizes ranging from 0.1 to 0.22 μm. Solutions of various characteristicsare optionally filtered using different filter membranes. For example,PVF and PTFE membranes are well suited to filtering organic solventswhile aqueous solutions are filtered through PVF or MCE membranes.Filter apparatus are available for use on many scales ranging from thesingle point-of-use disposable filter attached to a syringe up tocommercial scale filters for use in manufacturing plants. The membranefilters are sterilized by autoclave or chemical sterilization.Validation of membrane filtration systems is performed followingstandardized protocols (Microbiological Evaluation of Filters forSterilizing Liquids, Vol 4, No. 3. Washington, D.C.: Health IndustryManufacturers Association, 1981) and involve challenging the membranefilter with a known quantity (ca. 10^(7/) cm²) of unusually smallmicroorganisms, such as Brevundimonas diminuta (ATCC 19146).

Pharmaceutical compositions are optionally sterilized by passing throughmembrane filters. Formulations comprising nanoparticles (U.S. Pat. No.6,139,870) or multilamellar vesicles (Richard et al., InternationalJournal of Pharmaceutics (2006), 312(1-2):144-50) are amenable tosterilization by filtration through 0.22 μm filters without destroyingtheir organized structure.

In some embodiments, the methods disclosed herein comprise sterilizingthe formulation (or components thereof) by means of filtrationsterilization. In another embodiment the auris-acceptable otictherapeutic agent formulation comprises a particle wherein the particleformulation is suitable for filtration sterilization. In a furtherembodiment said particle formulation comprises particles of less than300 nm in size, of less than 200 nm in size, of less than 100 nm insize. In another embodiment the auris-acceptable formulation comprises aparticle formulation wherein the sterility of the particle is ensured bysterile filtration of the precursor component solutions. In anotherembodiment the auris-acceptable formulation comprises a particleformulation wherein the sterility of the particle formulation is ensuredby low temperature sterile filtration. In a further embodiment, lowtemperature sterile filtration is carried out at a temperature between 0and 30° C., between 0 and 20° C., between 0 and 10° C., between 10 and20° C., or between 20 and 30° C.

In another embodiment is a process for the preparation of anauris-acceptable particle formulation comprising: filtering the aqueoussolution containing the particle formulation at low temperature througha sterilization filter; lyophilizing the sterile solution; andreconstituting the particle formulation with sterile water prior toadministration. In some embodiments, a formulation described herein ismanufactured as a suspension in a single vial formulation containing themicronized active pharmaceutical ingredient. A single vial formulationis prepared by aseptically mixing a sterile poloxamer solution withsterile micronized active ingredient (e.g., thalidomide) andtransferring the formulation to sterile pharmaceutical containers. Insome embodiments, a single vial containing a formulation describedherein as a suspension is resuspended before dispensing and/oradministration.

In specific embodiments, filtration and/or filling procedures arecarried out at about 5° C. below the gel temperature (Tgel) of aformulation described herein and with viscosity below a theoreticalvalue of 100 cP to allow for filtration in a reasonable time using aperistaltic pump.

In another embodiment the auris-acceptable otic therapeutic agentformulation comprises a nanoparticle formulation wherein thenanoparticle formulation is suitable for filtration sterilization. In afurther embodiment the nanoparticle formulation comprises nanoparticlesof less than 300 nm in size, of less than 200 nm in size, or of lessthan 100 nm in size. In another embodiment the auris-acceptableformulation comprises a microsphere formulation wherein the sterility ofthe microsphere is ensured by sterile filtration of the precursororganic solution and aqueous solutions. In another embodiment theauris-acceptable formulation comprises a thermoreversible gelformulation wherein the sterility of the gel formulation is ensured bylow temperature sterile filtration. In a further embodiment, the lowtemperature sterile filtration occurs at a temperature between 0 and 30°C., or between 0 and 20° C., or between 0 and 10° C., or between 10 and20° C., or between 20 and 30° C. In another embodiment is a process forthe preparation of an auris-acceptable thermoreversible gel formulationcomprising: filtering the aqueous solution containing thethermoreversible gel components at low temperature through asterilization filter; lyophilizing the sterile solution; andreconstituting the thermoreversible gel formulation with sterile waterprior to administration.

In certain embodiments, the active ingredients are dissolved in asuitable vehicle (e.g. a buffer) and sterilized separately (e.g. by heattreatment, filtration, gamma radiation). In some instances, the activeingredients are sterilized separately in a dry state. In some instances,the active ingredients are sterilized as a suspension or as a colloidalsuspension. The remaining excipients (e.g., fluid gel components presentin auris formulations) are sterilized in a separate step by a suitablemethod (e.g. filtration and/or irradiation of a cooled mixture ofexcipients); the two solutions that are separately sterilized are thenmixed aseptically to provide a final auris formulation. In someinstances, the final aseptic mixing is performed just prior toadministration of a formulation described herein.

In some instances, conventionally used methods of sterilization (e.g.,heat treatment (e.g., in an autoclave), gamma irradiation, filtration)lead to irreversible degradation of polymeric components (e.g.,thermosetting, gelling or mucoadhesive polymer components) and/or theactive agent in the formulation. In some instances, sterilization of anauris formulation by filtration through membranes (e.g., 0.2 μMmembranes) is not possible if the formulation comprises thixotropicpolymers that gel during the process of filtration.

Accordingly, provided herein are methods for sterilization of aurisformulations that prevent degradation of polymeric components (e.g.,thermosetting and/or gelling and/or mucoadhesive polymer components)and/or the active agent during the process of sterilization. In someembodiments, degradation of the active agent (e.g., any therapeutic oticagent described herein) is reduced or eliminated through the use ofspecific pH ranges for buffer components and specific proportions ofgelling agents in the formulations. In some embodiments, the choice ofan appropriate gelling agent and/or thermosetting polymer allows forsterilization of formulations described herein by filtration. In someembodiments, the use of an appropriate thermosetting polymer and anappropriate copolymer (e.g., a gelling agent) in combination with aspecific pH range for the formulation allows for high temperaturesterilization of formulations described with substantially nodegradation of the therapeutic agent or the polymeric excipients. Anadvantage of the methods of sterilization provided herein is that, incertain instances, the formulations are subjected to terminalsterilization via autoclaving without any loss of the active agentand/or excipients and/or polymeric components during the sterilizationstep and are rendered substantially free of microbes and/or pyrogens.

Microorganisms

Provided herein are auris-acceptable compositions or devices thatameliorate or lessen otic disorders described herein. Further providedherein are methods comprising the administration of said oticcompositions. In some embodiments, the compositions or devices aresubstantially free of microorganisms. Acceptable bioburden or sterilitylevels are based on applicable standards that define therapeuticallyacceptable compositions, including but not limited to United StatesPharmacopeia Chapters <1111> et seq. For example, acceptable sterility(e.g., bioburden) levels include about 10 colony forming units (cfu) pergram of formulation, about 50 cfu per gram of formulation, about 100 cfuper gram of formulation, about 500 cfu per gram of formulation or about1000 cfu per gram of formulation. In some embodiments, acceptablebioburden levels or sterility for formulations include less than 10cfu/mL, less that 50 cfu/mL, less than 500 cfu/mL or less than 1000cfu/mL microbial agents. In addition, acceptable bioburden levels orsterility include the exclusion of specified objectionablemicrobiological agents. By way of example, specified objectionablemicrobiological agents include but are not limited to Escherichia coli(E. coli), Salmonella sp., Pseudomonas aeruginosa (P. aeruginosa) and/orother specific microbial agents.

Sterility of the auris-acceptable otic therapeutic agent formulation isconfirmed through a sterility assurance program in accordance withUnited States Pharmacopeia Chapters <61>, <62> and <71>. A key componentof the sterility assurance quality control, quality assurance andvalidation process is the method of sterility testing. Sterilitytesting, by way of example only, is performed by two methods. The firstis direct inoculation wherein a sample of the composition to be testedis added to growth medium and incubated for a period of time up to 21days. Turbidity of the growth medium indicates contamination. Drawbacksto this method include the small sampling size of bulk materials whichreduces sensitivity, and detection of microorganism growth based on avisual observation. An alternative method is membrane filtrationsterility testing. In this method, a volume of product is passed througha small membrane filter paper. The filter paper is then placed intomedia to promote the growth of microorganisms. This method has theadvantage of greater sensitivity as the entire bulk product is sampled.The commercially available Millipore Steritest sterility testing systemis optionally used for determinations by membrane filtration sterilitytesting. For the filtration testing of creams or ointments Steritestfilter system No. TLHVSL210 are used. For the filtration testing ofemulsions or viscous products Steritest filter system No. TLAREM210 orTDAREM210 are used. For the filtration testing of pre-filled syringesSteritest filter system No. TTHASY210 are used. For the filtrationtesting of material dispensed as an aerosol or foam Steritest filtersystem No. TTHVA210 are used. For the filtration testing of solublepowders in ampoules or vials Steritest filter system No. TTHADA210 orTTHADV210 are used.

Testing for E. coli and Salmonella includes the use of lactose brothsincubated at 30-35° C. for 24-72 hours, incubation in MacConkey and/orEMB agars for 18-24 hours, and/or the use of Rappaport medium. Testingfor the detection of P. aeruginosa includes the use of NAC agar. UnitedStates Pharmacopeia Chapter <62> further enumerates testing proceduresfor specified objectionable microorganisms.

In certain embodiments, any controlled release formulation describedherein has less than about 60 colony forming units (CFU), less thanabout 50 colony forming units, less than about 40 colony forming units,or less than about 30 colony forming units of microbial agents per gramof formulation. In certain embodiments, the otic formulations describedherein are formulated to be isotonic with the endolymph and/or theperilymph.

Endotoxins

Provided herein are otic compositions that ameliorate or lessen oticdisorders described herein. Further provided herein are methodscomprising the administration of said otic compositions. In someembodiments, the compositions or devices are substantially free ofendotoxins. An additional aspect of the sterilization process is theremoval of by-products from the killing of microorganisms (hereinafter,“Product”). The process of depyrogenation removes pyrogens from thesample. Pyrogens are endotoxins or exotoxins which induce an immuneresponse. An example of an endotoxin is the lipopolysaccharide (LPS)molecule found in the cell wall of gram-negative bacteria. Whilesterilization procedures such as autoclaving or treatment with ethyleneoxide kill the bacteria, the LPS residue induces a proinflammatoryimmune response, such as septic shock. Because the molecular size ofendotoxins can vary widely, the presence of endotoxins is expressed in“endotoxin units” (EU). One EU is equivalent to 100 picograms of E. coliLPS. Humans can develop a response to as little as 5 EU/kg of bodyweight. The bioburden (e.g., microbial limit) and/or sterility (e.g.,endotoxin level) is expressed in any units as recognized in the art. Incertain embodiments, otic compositions described herein contain lowerendotoxin levels (e.g. <4 EU/kg of body weight of a subject) whencompared to conventionally acceptable endotoxin levels (e.g., 5 EU/kg ofbody weight of a subject). In some embodiments, the auris-acceptableotic therapeutic agent formulation has less than about 5 EU/kg of bodyweight of a subject. In other embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 4 EU/kg of body weightof a subject. In additional embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 3 EU/kg of body weightof a subject. In additional embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 2 EU/kg of body weightof a subject.

In some embodiments, the auris-acceptable otic therapeutic agentformulation or device has less than about 5 EU/kg of formulation. Inother embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 4 EU/kg of formulation. In additionalembodiments, the auris-acceptable otic therapeutic agent formulation hasless than about 3 EU/kg of formulation. In some embodiments, theauris-acceptable otic therapeutic agent formulation has less than about5 EU/kg Product. In other embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 1 EU/kg Product. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 0.2 EU/kg Product. In some embodiments,the auris-acceptable otic therapeutic agent formulation has less thanabout 5 EU/g of unit or Product. In other embodiments, theauris-acceptable otic therapeutic agent formulation has less than about4 EU/g of unit or Product. In additional embodiments, theauris-acceptable otic therapeutic agent formulation has less than about3 EU/g of unit or Product. In some embodiments, the auris-acceptableotic therapeutic agent formulation has less than about 5 EU/mg of unitor Product. In other embodiments, the auris-acceptable otic therapeuticagent formulation has less than about 4 EU/mg of unit or Product. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 3 EU/mg of unit or Product. In certainembodiments, otic compositions described herein contain from about 1 toabout 5 EU/mL of formulation. In certain embodiments, otic compositionsdescribed herein contain from about 2 to about 5 EU/mL of formulation,from about 3 to about 5 EU/mL of formulation, or from about 4 to about 5EU/mL of formulation.

In certain embodiments, otic compositions or devices described hereincontain lower endotoxin levels (e.g. <0.5 EU/mL of formulation) whencompared to conventionally acceptable endotoxin levels (e.g., 0.5 EU/mLof formulation). In some embodiments, the auris-acceptable otictherapeutic agent formulation or device has less than about 0.5 EU/mL offormulation. In other embodiments, the auris-acceptable otic therapeuticagent formulation has less than about 0.4 EU/mL of formulation. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 0.2 EU/mL of formulation.

Pyrogen detection, by way of example only, is performed by severalmethods. Suitable tests for sterility include tests described in UnitedStates Pharmacopoeia (USP) <71> Sterility Tests (23rd edition, 1995).The rabbit pyrogen test and the Limulus amebocyte lysate test are bothspecified in the United States Pharmacopeia Chapters <85> and <151>(USP23/NF 18, Biological Tests, The United States PharmacopeialConvention, Rockville, Md., 1995). Alternative pyrogen assays have beendeveloped based upon the monocyte activation-cytokine assay. Uniformcell lines suitable for quality control applications have been developedand have demonstrated the ability to detect pyrogenicity in samples thathave passed the rabbit pyrogen test and the Limulus amebocyte lysatetest (Taktak et al, J. Pharm. Pharmacol. (1990), 43:578-82). In anadditional embodiment, the auris-acceptable otic therapeutic agentformulation is subject to depyrogenation. In a further embodiment, theprocess for the manufacture of the auris-acceptable otic therapeuticagent formulation comprises testing the formulation for pyrogenicity. Incertain embodiments, the formulations described herein are substantiallyfree of pyrogens.

pH and Practical Osmolarity

As used herein, “practical osmolarity” means the osmolarity of aformulation that is measured by including the active agent and allexcipients except the gelling and/or the thickening agent (e.g.,polyoxyethylene-polyooxypropylene copolymers, carboxymethylcellulose orthe like). The practical osmolarity of a formulation described herein ismeasured by any suitable method, e.g., a freezing point depressionmethod as described in Viegas et. al., Int. J. Pharm., 1998, 160,157-162. In some instances, the practical osmolarity of a compositiondescribed herein is measured by vapor pressure osmometry (e.g., vaporpressure depression method) that allows for determination of theosmolarity of a composition at higher temperatures. In some instances,vapor pressure depression method allows for determination of theosmolarity of a formulation comprising a gelling agent (e.g., athermoreversible polymer) at a higher temperature wherein the gellingagent is in the form of a gel. The practical osmolality of an oticformulation described herein is from about 100 mOsm/kg to about 1000mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about280 mOsm/kg to about 320 mOsm/kg. In some embodiments, the formulationsdescribed herein have a practical osmolarity of about 100 mOsm/L toabout 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L.

In some embodiments, the osmolarity at a target site of action (e.g.,the perilymph) is about the same as the delivered osmolarity (i.e.,osmolarity of materials that cross or penetrate the round windowmembrane) of any formulation described herein. In some embodiments, theformulations described herein have a delieverable osmolarity of about150 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 500 mOsm/L,about 250 mOsm/L to about 350 mOsm/L, about 280 mOsm/L to about 370mOsm/L or about 250 mOsm/L to about 320 mOsm/L.

The main cation present in the endolymph is potassium. In addition theendolymph has a high concentration of positively charged amino acids.The main cation present in the perilymph is sodium. In certaininstances, the ionic composition of the endolymph and perilymph regulatethe electrochemical impulses of hair cells. In certain instances, anychange in the ionic balance of the endolymph or perilymph results in aloss of hearing due to changes in the conduction of electrochemicalimpulses along otic hair cells. In some embodiments, a compositiondisclosed herein does not disrupt the ionic balance of the perilymph. Insome embodiments, a composition disclosed herein has an ionic balancethat is the same as or substantially the same as the perilymph. In someembodiments, a composition disclosed herein does not disrupt the ionicbalance of the endolymph. In some embodiments, a composition disclosedherein has an ionic balance that is the same as or substantially thesame as the endolymph. In some embodiments, an otic formulationdescribed herein is formulated to provide an ionic balance that iscompatible with inner ear fluids (e.g., endolymph and/or perilymph).

The endolymph and the perilymph have a pH that is close to thephysiological pH of blood. The endolymph has a pH range of about7.2-7.9; the perilymph has a pH range of about 7.2-7.4. The in situ pHof the proximal endolymph is about 7.4 while the pH of distal endolymphis about 7.9.

In some embodiments, the pH of a composition described herein isadjusted (e.g., by use of a buffer) to an endolymph-compatible pH rangeof about 5.5 to 9.0. In specific embodiments, the pH of a compositiondescribed herein is adjusted to a perilymph-suitable pH range of about5.5 to about 9.0. In some embodiments, the pH of a composition describedherein is adjusted to a perilymph-suitable range of about 5.5 to about8.0, about 6 to about 8.0 or about 6.6 to about 8.0. In someembodiments, the pH of a composition described herein is adjusted to aperilymph-suitable pH range of about 7.0-7.6.

In some embodiments, useful formulations also include one or more pHadjusting agents or buffering agents. Suitable pH adjusting agents orbuffers include, but are not limited to acetate, bicarbonate, ammoniumchloride, citrate, phosphate, pharmaceutically acceptable salts thereofand combinations or mixtures thereof.

In one embodiment, when one or more buffers are utilized in theformulations of the present disclosure, they are combined, e.g., with apharmaceutically acceptable vehicle and are present in the finalformulation, e.g., in an amount ranging from about 0.1% to about 20%,from about 0.5% to about 10%. In certain embodiments of the presentdisclosure, the amount of buffer included in the gel formulations are anamount such that the pH of the gel formulation does not interfere withthe body's natural buffering system.

In one embodiment, diluents are also used to stabilize compounds becausethey can provide a more stable environment. Salts dissolved in bufferedsolutions (which also can provide pH control or maintenance) areutilized as diluents in the art, including, but not limited to aphosphate buffered saline solution.

In some embodiments, any gel formulation described herein has a pH thatallows for sterilization (e.g., by filtration or aseptic mixing or heattreatment and/or autoclaving (e.g., terminal sterilization) of a gelformulation without degradation of the pharmaceutical agent (e.g.,cytotoxic agent) or the polymers comprising the gel. In order to reducehydrolysis and/or degradation of the otic agent and/or the gel polymerduring sterilization, the buffer pH is designed to maintain pH of theformulation in the 7-8 range during the process of sterilization (e.g.,high temperature autoclaving).

In specific embodiments, any gel formulation described herein has a pHthat allows for terminal sterilization (e.g., by heat treatment and/orautoclaving) of a gel formulation without degradation of thepharmaceutical agent (e.g., cytotoxic agent) or the polymers comprisingthe gel. For example, in order to reduce hydrolysis and/or degradationof the otic agent and/or the gel polymer during autoclaving, the bufferpH is designed to maintain pH of the formulation in the 7-8 range atelevated temperatures. Any appropriate buffer is used depending on theotic agent used in the formulation. In some instances, since pK_(a) ofTRIS decreases as temperature increases at approximately −0.03/° C. andpK_(a) of PBS increases as temperature increases at approximately0.003/° C., autoclaving at 250° F. (121° C.) results in a significantdownward pH shift (i.e. more acidic) in the TRIS buffer whereas arelatively much less upward pH shift in the PBS buffer and thereforemuch increased hydrolysis and/or degradation of an otic agent in TRISthan in PBS. Degradation of an otic agent is reduced by the use of anappropriate combination of a buffer and polymeric additives (e.g. CMC)as described herein.

In some embodiments, a formulation pH of between about 5.0 and about9.0, between about 5.5 and about 8.5, between about 6.0 and about 7.6,between about 7 and about 7.8, between about 7.0 and about 7.6, betweenabout 7.2 and 7.6, or between about 7.2 and about 7.4 is suitable forsterilization (e.g., by filtration or aseptic mixing or heat treatmentand/or autoclaving (e.g., terminal sterilization)) of auris formulationsdescribed herein. In specific embodiments a formulation pH of about 6.0,about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about7.5, or about 7.6 is suitable for sterilization (e.g., by filtration oraseptic mixing or heat treatment and/or autoclaving (e.g., terminalsterilization)) of any composition described herein.

In some embodiments, the formulations have a pH as described herein, andinclude a thickening agent (e.g., a vicosity enhancing agent) such as,by way of non-limiting example, a cellulose based thickening agentdescribed herein. In some instances, the addition of a secondary polymer(e.g., a thickening agent) and a pH of formulation as described herein,allows for sterilization of a formulation described herein without anysubstantial degradation of the otic agent and/or the polymer componentsin the otic formulation. In some embodiments, the ratio of athermoreversible poloxamer to a thickening agent in a formulation thathas a pH as described herein, is about 40:1, about 35:1, about 30:1,about 25:1, about 20:1, about 15:1 about 10:l, or about 5:1. Forexample, in certain embodiments, a sustained and/or extended releaseformulation described herein comprises a combination of poloxamer 407(pluronic F127) and carboxymethylcellulose (CMC) in a ratio of about40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1, about10:1 or about 5:1.

In some embodiments, the amount of thermoreversible polymer in anyformulation described herein is about 10%, about 15%, about 20%, about25%, about 30%, about 35% or about 40% of the total weight of theformulation. In some embodiments, the amount of thermoreversible polymerin any formulation described herein is about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24% or about 25%of the total weight of the formulation. In some embodiments, the amountof thermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 7.5% of the total weight of the formulation.In some embodiments, the amount of thermoreversible polymer (e.g.,pluronic F127) in any formulation described herein is about 10% of thetotal weight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 11% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 12% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 13% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 14% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 15% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 16% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 17% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 18% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 19% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 20% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 21% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 23% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 25% of the total weight of the formulation.

In some embodiments, the amount of thickening agent (e.g., a gellingagent) in any formulation described herein is about 1%, about 5%, about10%, or about 15% of the total weight of the formulation. In someembodiments, the amount of thickening agent (e.g., a gelling agent) inany formulation described herein is about 0.5%, about 1%, about 1.5%,about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, orabout 5% of the total weight of the formulation.

In some embodiments, the pharmaceutical formulations described hereinare stable with respect to pH over a period of any of at least about 1day, at least about 2 days, at least about 3 days, at least about 4days, at least about 5 days, at least about 6 days, at least about 1week, at least about 2 weeks, at least about 3 weeks, at least about 4weeks, at least about 5 weeks, at least about 6 weeks, at least about0.7 weeks, at least about 8 weeks, at least about 1 month, at leastabout 2 months, at least about 3 months, at least about 4 months, atleast about 5 months, or at least about 6 months. In other embodiments,the formulations described herein are stable with respect to pH over aperiod of at least about 1 week. Also described herein are formulationsthat are stable with respect to pH over a period of at least about 1month.

Tonicity Agents

In general, the endolymph has a higher osmolality than the perilymph.For example, the endolymph has an osmolality of about 304 mOsm/kg H₂Owhile the perilymph has an osmolality of about 294 mOsm/kg H₂O. Incertain embodiments, tonicity agents are added to the formulationsdescribed herein in an amount as to provide a practical osmolality of anotic formulation of about 100 mOsm/kg to about 1000 mOsm/kg, from about200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about280 mOsm/kg to about 320 mOsm/kg. In some embodiments, the formulationsdescribed herein have a practical osmolarity of about 100 mOsm/L toabout 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about280 mOsm/L to about 320 mOsm/L or about 250 mOsm/L to about 320 mOsm/L.

In some embodiments, the deliverable osmolarity of any formulationdescribed herein is designed to be isotonic with the targeted oticstructure (e.g., endolymph, perilymph or the like). In specificembodiments, auris compositions described herein are formulated toprovide a delivered perilymph-suitable osmolarity at the target site ofaction of about 250 to about 320 mOsm/L; and preferably about 270 toabout 320 mOsm/L. In specific embodiments, auris compositions describedherein are formulated to provide a delivered perilymph-suitableosmolality at the target site of action of about 250 to about 320mOsm/kg H₂O; or an osmolality of about 270 to about 320 mOsm/kg H₂O. Inspecific embodiments, the deliverable osmolarity/osmolality of theformulations (i.e., the osmolarity/osmolality of the formulation in theabsence of gelling or thickening agents (e.g., thermoreversible gelpolymers) is adjusted, for example, by the use of appropriate saltconcentrations (e.g., concentration of potassium or sodium salts) or theuse of tonicity agents which renders the formulationsendolymph-compatible and/or perilymph-compatible (i.e. isotonic with theendolymph and/or perilymph) upon delivery at the target site. Theosmolarity of a formulation comprising a thermoreversible gel polymer isan unreliable measure due to the association of varying amounts of waterwith the monomeric units of the polymer. The practical osmolarity of aformulation (i.e., osmolarity in the absence of a gelling or thickeningagent (e.g. a thermoreversible gel polymer) is a reliable measure and ismeasured by any suitable method (e.g., freezing point depression method,vapor depression method). In some instances, the formulations describedherein provide a deliverable osmolarity (e.g., at a target site (e.g.,perilymph) that causes minimal disturbance to the environment of theinner ear and causes minimum discomfort (e.g., vertigo and/or nausea) toa mammal upon administration.

In some embodiments, any formulation described herein is isotonic withthe perilymph and/or endolymph. Isotonic formulations are provided bythe addition of a tonicity agent. Suitable tonicity agents include, butare not limited to any pharmaceutically acceptable sugar, salt or anycombinations or mixtures thereof, such as, but not limited to dextrose,glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

Useful auris compositions include one or more salts in an amountrequired to bring osmolality of the composition into an acceptablerange. Such salts include those having sodium, potassium or ammoniumcations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some embodiments, the formulations described herein have a pH and/orpractical osmolarity as described herein, and have a concentration ofactive pharmaceutical ingredient between about 1 μM and about 10 μM,between about 1 mM and about 100 mM, between about 0.1 mM and about 100mM, between about 0.1 mM and about 100 nM. In some embodiments, theformulations described herein have a pH and/or practical osmolarity asdescribed herein, and have a concentration of active pharmaceuticalingredient between about 0.01%-about 20%, between about 0.01%-about 10%,between about 0.01%-about 7.5%, between about 0.01%-6%, between about0.01-5%, between about 0.1-about 10%, or between about 0.1-about 6% ofthe active ingredient by weight of the formulation. In some embodiments,the formulations described herein have a pH and/or practical osmolarityas described herein, and have a concentration of active pharmaceuticalingredient between about 0.1 and about 70 mg, between about 1 mg andabout 70 mg/mL, between about 1 mg and about 50 mg/mL, between about 1mg/mL and about 20 mg/mL, between about 1 mg/mL to about 10 mg/mL,between about 1 mg/mL to about 5 mg/mL, or between about 0.5 mg/mL toabout 5 mg/mL of the active agent by volume of the formulation. In someembodiments, the formulations described herein have a pH and/orpractical osmolarity as described herein, and have a concentration ofactive pharmaceutical ingredient between about 1 μg/mL and about 500μg/mL, between about 1 μg/mL and about 250 μg/mL, between about 1 μg andabout 100 μg/mL, between about 1 μg/mL and about 501 g/mL, or betweenabout 1 μg/mL and about 20 μg/mL of the active agent by volume of theformulation.

Particle Size

Size reduction is used to increase surface area and/or modulateformulation dissolution properties. It is also used to maintain aconsistent average particle size distribution (PSD) (e.g.,micrometer-sized particles, nanometer-sized particles or the like) forany formulation described herein. In some embodiments, any formulationdescribed herein comprises multiparticulates, i.e., a plurality ofparticle sizes (e.g., micronized particles, nano-sized particles,non-sized particles, colloidal particles); i.e., the formulation is amultiparticulate formulation. In some embodiments, any formulationdescribed herein comprises one or more multiparticulate (e.g.,micronized) therapeutic agents. Micronization is a process of reducingthe average diameter of particles of a solid material. Micronizedparticles are from about micrometer-sized in diameter to aboutnanometer-sized in diameter. In some embodiments, the average diameterof particles in a micronized solid is from about 0.5 μm to about 500 μm.In some embodiments, the average diameter of particles in a micronizedsolid is from about 1 μm to about 200 μm. In some embodiments, theaverage diameter of particles in a micronized solid is from about 2 μmto about 100 μm. In some embodiments, the average diameter of particlesin a micronized solid is from about 3 μm to about 50 μm. In someembodiments, a particulate micronized solid comprises particle sizes ofless than about 5 microns, less than about 20 microns and/or less thanabout 100 microns. In some embodiments, the use of particulates (e.g.,micronized particles) of cytotoxic agent allows for extended and/orsustained release of the cytotoxic agent from any formulation describedherein compared to a formulation comprising non-multiparticulate (e.g.,non-micronized) cytotoxic agent. In some instances, formulationscontaining multiparticulate (e.g. micronized) cytotoxic agent areejected from a 1 mL syringe adapted with a 27 G needle without anyplugging or clogging.

In some instances, any particle in any formulation described herein is acoated particle (e.g., a coated micronized particle, nano-particle)and/or a microsphere and/or a liposomal particle. Particle sizereduction techniques include, by way of example, grinding, milling(e.g., air-attrition milling (jet milling), ball milling), coacervation,complex coacervation, high pressure homogenization, spray drying and/orsupercritical fluid crystallization. In some instances, particles aresized by mechanical impact (e.g., by hammer mills, ball mill and/or pinmills). In some instances, particles are sized via fluid energy (e.g.,by spiral jet mills, loop jet mills, and/or fluidized bed jet mills). Insome embodiments formulations described herein comprise crystallineparticles and/or isotropic particles. In some embodiments, formulationsdescribed herein comprise amorphous particles and/or anisotropicparticles. In some embodiments, formulations described herein comprisetherapeutic agent particles wherein the therapeutic agent is a freebase, or a salt, or a prodrug of a therapeutic agent, or any combinationthereof.

In some embodiments, a formulation described herein comprises one ormore cytotoxic agents wherein the cytotoxic agent comprisesnanoparticulates. In some embodiments, a formulation described hereincomprises cytotoxic agent beads (e.g., cyclophosphamide beads) that areoptionally coated with controlled release excipients. In someembodiments, a formulation described herein comprises a cytotoxicagentthat is granulated and/or reduced in size and coated withcontrolled release excipients; the granulated coated cytotoxicagentparticulates are then optionally micronized and/or formulated inany of the compositions described herein.

In some instances, a combination of a cytotoxic agent as a neutralmolecule, free acid or free base and a salt of the cytotoxic agent isused to prepare pulsed release otic agent formulations using theprocedures described herein. In some formulations, a combination of amicronized cytotoxic agent (and/or salt or prodrug thereof) and coatedparticles (e.g., nanoparticles, liposomes, microspheres) is used toprepare pulsed release otic agent formulations using any proceduredescribed herein. Altemtaively, a pulsed release profile is achieved bysolubilizing up to 20% of the delivered dose of the cytotoxic agent(e.g., micronized cytotoxic agent, free base, free acid or salt orprodrug thereof; multiparticulate cytotoxic agent, free base, free acidor salt or prodrug thereof) with the aid of cyclodextrins, surfactants(e.g., poloxamers (407, 338, 188), tween (80, 60, 20, 81),PEG-hydrogenated castor oil, cosolvents like N-methyl-2-Pyrrolidone orthe like and preparing pulsed release formulations using any proceduredescribed herein.

In specific embodiments, any auris-compatible formulation describedherein comprises one or more micronized pharmaceutical agents (e.g.,cytotoxic agents). In some of such embodiments, a micronizedpharmaceutical agent comprises micronized particles, coated (e.g., withan extended release coat) micronized particles, or a combinationthereof. In some of such embodiments, a micronized pharmaceutical agentcomprising micronized particles, coated micronized particles, or acombination thereof, comprises a cytotoxic agent as a neutral molecule,a free acid, a free base, a salt, a prodrug or any combination thereof.In certain embodiments, a pharmaceutical composition described hereincomprises a cytotoxic agent as a micronized powder. In certainembodiments, a pharmaceutical composition described herein comprises acytotoxic agent in the form of a micro-cytotoxic agent powder.

The multiparticulates and/or micronized cytotoxic agents describedherein are delivered to an auris structure (e.g., inner ear) by means ofany type of matrix including solid, liquid or gel matrices. In someembodiments, the multiparticulates and/or micronized cytotoxic agentsdescribed herein are delivered to an auris structure (e.g., inner ear)by means of any type of matrix including solid, liquid or gel matricesvia intratympanic injection.

Pharmaceutical Formulations

Provided herein are pharmaceutical compositions or devices that includeat least one cytotoxic agent and a pharmaceutically acceptablediluent(s), excipient(s), or carrier(s). In some embodiments, thepharmaceutical compositions include other medicinal or pharmaceuticalagents, carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In other embodiments, the pharmaceuticalcompositions also contain other therapeutic substances.

In some embodiments, the compositions or devices described hereininclude a dye to help enhance the visualization of the gel when applied.In some embodiments, dyes that are compatible with the auris-acceptablecompositions or devices described herein include Evans blue (e.g., 0.5%of the total weight of an otic formulation), Methylene blue (e.g., 1% ofthe total weight of an otic formulation), Isosulfan blue (e.g., 1% ofthe total weight of an otic formulation), Trypan blue (e.g., 0.15% ofthe total weight of an otic formulation), and/or indocyanine green(e.g., 25 mg/vial). Other common dyes, e.g., FD&C red 40, FD&C red 3,FD&C yellow 5, FD&C yellow 6, FD&C blue 1, FD&C blue 2, FD&C green 3,fluorescence dyes (e.g., Fluorescein isothiocyanate, rhodamine, AlexaFluors, DyLight Fluors) and/or dyes that are visualizable in conjunctionwith non-invasive imaging techniques such as MRI, CAT scans, PET scansor the like. Gadolinium-based MRI dyes, iodine-base dyes, barium-baseddyes or the like are also contemplated for use with any otic formulationdescribed herein. Other dyes that are compatible with any formulation orcomposition described herein are listed in the Sigma-Aldrich catalogunder dyes (which is included herein by reference for such disclosure).

Any pharmaceutical composition or device described herein isadministered by locating the composition or device in contact with thecrista fenestrae cochlea, the round window, the tympanic cavity, thetympanic membrane, the auris media or the auris externa.

In one specific embodiment of the auris-acceptable controlled releasecytotoxic agent pharmaceutical formulations described herein, thecytotoxic agent is provided in a gel matrix, also referred to herein as“auris acceptable gel formulations,” “auris interna-acceptable gelformulations,” “auris media-acceptable gel formulations,” “aurisextema-acceptable gel formulations”, “auris gel formulations” orvariations thereof. All of the components of the gel formulation must becompatible with the targeted auris structure. Further, the gelformulations provide controlled release of the cytotoxic agent to thedesired site within the targeted auris structure; in some embodiments,the gel formulation also has an immediate or rapid release component fordelivery of the cytotoxic agent to the desired target site. In otherembodiments, the gel formulation has a sustained release component fordelivery of the cytotoxic agent. In some embodiments, the gelformulation comprises a multiparticulate (e.g., micronized) cytotoxicagent. In some embodiments, the auris gel formulations arebiodegradeable. In other embodiments, the auris gel formulations includea mucoadhesive excipient to allow adhesion to the external mucous layerof the round window membrane. In yet other embodiments, the auris gelformulations include a penetration enhancer excipient; in furtherembodiments, the auris gel formulation contains a viscosity enhancingagent sufficient to provide a viscosity of between about 500 and1,000,000 centipoise, between about 750 and 1,000,000 centipoise;between about 1000 and 1,000,000 centipoise; between about 1000 and400,000 centipoise; between about 2000 and 100,000 centipoise; betweenabout 3000 and 50,000 centipoise; between about 4000 and 25,000centipoise; between about 5000 and 20,000 centipoise; or between about6000 and 15,000 centipoise. In some embodiments, the auris gelformulation contains a viscosity enhancing agent sufficient to provide aviscosity of between about 50,0000 and 1,000,000 centipoise.

In other embodiments, the auris interna pharmaceutical formulationsdescribed herein further provide an auris-acceptable hydrogel; in yetother embodiments, the auris pharmaceutical formulations provide anauris-acceptable microsphere or microparticle; in still otherembodiments, the auris pharmaceutical formulations provide anauris-acceptable liposome. In some embodiments, the auris pharmaceuticalformulations provide an auris-acceptable foam; in yet other embodiments,the auris pharmaceutical formulations provide an auris-acceptable paint;in still further embodiments, the auris pharmaceutical formulationsprovide an auris-acceptable in situ forming spongy material. In someembodiments, the auris pharmaceutical formulations provide anauris-acceptable solvent release gel. In some embodiments, the aurispharmaceutical formulations provide an actinic radiation curable gel.Further embodiments include a thermoreversible gel in the aurispharmaceutical formulation, such that upon preparation of the gel atroom temperature or below, the formulation is a fluid, but uponapplication of the gel into or near the auris interna and/or auris mediatarget site, including the tympanic cavity, round window membrane or thecrista fenestrae cochleae, the auris-pharmaceutical formulation stiffensor hardens into a gel-like substance.

In further or alternative embodiments, the auris gel formulations arecapable of being administered on or near the round window membrane viaintratympanic injection. In other embodiments, the auris gelformulations are administered on or near the round window or the cristafenestrae cochleae through entry via a post-auricular incision andsurgical manipulation into or near the round window or the cristafenestrae cochleae area. Alternatively, the auris gel formulation isapplied via syringe and needle, wherein the needle is inserted throughthe tympanic membrane and guided to the area of the round window orcrista fenestrae cochleae. The auris gel formulations are then depositedon or near the round window or crista fenestrae cochleae for localizedtreatment of autoimmune otic disorders. In other embodiments, the aurisgel formulations are applied via microcathethers implanted into thepatient, and in yet further embodiments the formulations areadministered via a pump device onto or near the round window membrane.In still further embodiments, the auris gel formulations are applied ator near the round window membrane via a microinjection device. In yetother embodiments, the auris gel formulations are applied in thetympanic cavity. In some embodiments, the auris gel formulations areapplied on the tympanic membrane. In still other embodiments, the aurisgel formulations are applied onto or in the auditory canal.

In further specific embodiments, any pharmaceutical composition ordevice described herein comprises a multiparticulate cytotoxic agent ina liquid matrix (e.g., a liquid composition for intratympanic injection,or otic drops). In certain embodiments, any pharmaceutical compositiondescribed herein comprises a multiparticulate cytotoxic agent in a solidmatrix.

Controlled Release Formulations

In general, controlled release drug formulations impart control over therelease of drug with respect to site of release and time of releasewithin the body. As discussed herein, controlled release refers toimmediate release, delayed release, sustained release, extended release,variable release, pulsatile release and bi-modal release. Manyadvantages are offered by controlled release. First, controlled releaseof a pharmaceutical agent allows less frequent dosing and thus minimizesrepeated treatment. Second, controlled release treatment results in moreefficient drug utilization and less of the compound remains as aresidue. Third, controlled release offers the possibility of localizeddrug delivery by placement of a delivery device or formulation at thesite of disease. Still further, controlled release offers theopportunity to administer and release two or more different drugs, eachhaving a unique release profile, or to release the same drug atdifferent rates or for different durations, by means of a single dosageunit.

Accordingly, one aspect of the embodiments disclosed herein is toprovide a controlled release cytotoxic agent auris-acceptablecomposition or device for the treatment of autoimmune disorders and/orinflammatory disorders. The controlled release aspect of thecompositions and/or formulations and/or devices disclosed herein isimparted through a variety of agents, including but not limited toexcipients, agents or materials that are acceptable for use in the aurisinterna or other otic structure. By way of example only, suchexcipients, agents or materials include an auris-acceptable polymer, anauris-acceptable viscosity enhancing agent, an auris-acceptable gel, anauris-acceptable paint, an auris-acceptable foam, an auris-acceptablexerogel, an auris-acceptable microsphere or microparticle, anauris-acceptable hydrogel, an auris-acceptable in situ forming spongymaterial, an auris-acceptable actinic radiation curable gel, anauris-acceptable solvent release gel, an auris-acceptable liposome, anauris-acceptable nanocapsule or nanosphere, an auris-acceptablethermoreversible gel, or combinations thereof.

Tunable Release Characteristics

The release of active agent from any formulation, composition or devicedescribed herein is optionally tunable to the desired releasecharacteristics. In some embodiments, a composition described herein isa solution that is substantially free of gelling components. In suchinstances, the composition provides essentially immediate release of anactive agent. In some of such embodiments, the composition is useful inperfusion of otic structures, e.g., during surgery.

In some embodiments, a composition described herein is a solution thatis substantially free of gelling components and comprises micronizedotic agent (e.g., a cytotoxic agent). In some of such embodiments, thecomposition provides release of an active agent from about 2 days toabout 4 days. In some embodiments, a composition described hereincomprises a gelling agent (e.g., poloxamer 407) and provides release ofan active agent over a period of from about 1 day to about 3 days. Insome embodiments, a composition described herein comprises a gellingagent (e.g., poloxamer 407) and provides release of an active agent overa period of from about 1 day to about 5 days. In some embodiments, acomposition described herein comprises a gelling agent (e.g., poloxamer407) and provides release of an active agent over a period of from about2 days to about 7 days.

In some embodiments, a composition described herein comprises a gellingagent (e.g., poloxamer 407) in combination with micronized otic agentand provides extended sustained release over a longer period of time. Insome embodiments, a composition described herein comprises about 14-17%of a gelling agent (e.g., poloxamer 407) and micronized otic agent, andprovides extended sustained release over a period of from about 1 weekto about 3 weeks. In some embodiments, a composition described hereincomprises about 18-21% of a gelling agent (e.g., poloxamer 407) andmicronized otic agent, and provides extended sustained release over aperiod of from about 3 weeks to about 6 weeks.

Accordingly, the amount of gelling agent in a composition, and theparticle size of an otic agent are tunable to the desired releaseprofile of an otic agent from the composition.

In specific embodiments, compositions comprising micronized otic agentsprovide extended release over a longer period of time compared tocompositions comprising non-micronized otic agents. In specificembodiments, selection of an appropriate particle size of the activeagent (e.g., micronized active agent) in combination with the amount ofgelling agent in the composition provides tunable extended releasecharacteristics that allow for release of an active agent over a periodof hours, days, weeks or months.

Auris-Acceptable Gels

Gels, sometimes referred to as jellies, have been defined in variousways. For example, the United States Pharmacopoeia defines gels assemisolid systems consisting of either suspensions made up of smallinorganic particles or large organic molecules interpenetrated by aliquid. Gels include a single-phase or a two-phase system. Asingle-phase gel consists of organic macromolecules distributeduniformly throughout a liquid in such a manner that no apparentboundaries exist between the dispersed macromolecules and the liquid.Some single-phase gels are prepared from synthetic macromolecules (e.g.,carbomer) or from natural gums, (e.g., tragacanth). In some embodiments,single-phase gels are generally aqueous, but will also be made usingalcohols and oils. Two-phase gels consist of a network of small discreteparticles.

Gels can also be classified as being hydrophobic or hydrophilic. Incertain embodiments, the base of a hydrophobic gel consists of a liquidparaffin with polyethylene or fatty oils gelled with colloidal silica,or aluminum or zinc soaps. In contrast, the base of hydrophobic gelsusually consists of water, glycerol, or propylene glycol gelled with asuitable gelling agent (e.g., tragacanth, starch, cellulose derivatives,carboxyvinylpolymers, and magnesium-aluminum silicates). In certainembodiments, the rheology of the compositions or devices disclosedherein is pseudo plastic, plastic, thixotropic, or dilatant.

In one embodiment the enhanced viscosity auris-acceptable formulationdescribed herein is not a liquid at room temperature. In certainembodiments, the enhanced viscosity formulation is characterized by aphase transition between room temperture and body temperature (includingan individual with a serious fever, e.g., up to about 42° C.). In someembodiments, the phase transition occurs at 1° C. below bodytemperature, at 2° C. below body temperature, at 3° C. below bodytemperture, at 4° C. below body temperature, at 6° C. below bodytemperature, at 8° C. below body temperature, or at 10° C. below bodytemperature. In some embodiments, the phase transition occurs at about15° C. below body temperature, at about 20° C. below body temperature orat about 25° C. below body temperature. In specific embodiments, thegelation temperature (Tgel) of a formulation described herein is about20° C., about 25° C., or about 30° C. In certain embodiments, thegelation temperature (Tgel) of a formulation described herein is about35° C., or about 40° C. In one embodiment, administration of anyformulation described herein at about body temperature reduces orinhibits vertigo associated with intratympanic administration of oticformulations. Included within the definition of body temperature is thebody temperature of a healthy individual, or an unhealthy individual,including an individual with a fever (up to ˜42° C.). In someembodiments, the pharmaceutical compositions or devices described hereinare liquids at about room temperature and are administered at or aboutroom temperature, reducing or ameliorating side effects such as, forexample, vertigo.

Polymers composed of polyoxypropylene and polyoxyethylene formthermoreversible gels when incorporated into aqueous solutions. Thesepolymers have the ability to change from the liquid state to the gelstate at tempertures close to body temperture, therefore allowing usefulformulations that are applied to the targeted auris structure(s). Theliquid state-to-gel state phase transition is dependent on the polymerconcentration and the ingredients in the solution.

Poloxamer 407 (PF-127) is a nonionic surfactant composed ofpolyoxyethylene-polyoxypropylene copolymers. Other poloxamers include188 (F-68 grade), 237 (F-87 grade), 338 (F-108 grade). Aqueous solutionsof poloxamers are stable in the presence of acids, alkalis, and metalions. PF-127 is a commercially availablepolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106, with an average molar mass of 13,000. The polymer can befurther purified by suitable methods that will enhance gelationproperties of the polymer. It contains approximately 70% ethylene oxide,which accounts for its hydrophilicity. It is one of the series ofpoloxamer ABA block copolymers, whose members share the chemical formulashown below.

PF-127 is of particular interest since concentrated solutions (>20% w/w)of the copolymer are transformed from low viscosity transparentsolutions to solid gels on heating to body temperature. This phenomenon,therefore, suggests that when placed in contact with the body, the gelpreparation will form a semi-solid structure and a sustained releasedepot. Furthermore, PF-127 has good solubilizing capacity, low toxicityand is, therefore, considered a good medium for drug delivery systems.

In an alternative embodiment, the thermogel is a PEG-PLGA-PEG triblockcopolymer (Jeong et al, Nature (1997), 388:860-2; Jeong et al, J.Control. Release (2000), 63:155-63; Jeong et al, Adv. Drug Delivery Rev.(2002), 54:37-51). The polymer exhibits sol-gel behavior over aconcentration of about 5% w/w to about 40% w/w. Depending on theproperties desired, the lactide/glycolide molar ratio in the PLGAcopolymer ranges from about 1:1 to about 20:1. The resulting coploymersare soluble in water and form a free-flowing liquid at room temperature,but form a hydrogel at body temperature. A commercially availablePEG-PLGA-PEG triblock copolymer is RESOMER RGP t50106 manufactured byBoehringer Ingelheim. This material is composed of a PGLA copolymer of50:50 poly(DL-lactide-co-glycolide) and is 10% w/w of PEG and has amolecular weight of about 6000.

ReGel® is a tradename of MacroMed Incorporated for a class of lowmolecular weight, biodegradable block copolymers having reverse thermalgelation properties as described in U.S. Pat. Nos. 6,004,573, 6,117949,6,201,072, and 6,287,588. It also includes biodegradable polymeric drugcarriers disclosed in pending U.S. patent application Ser. Nos.09/906,041, 09/559,799 and 10/919,603. The biodegradable drug carriercomprises ABA-type or BAB-type triblock copolymers or mixtures thereof,wherein the A-blocks are relatively hydrophobic and comprisebiodegradable polyesters or poly(orthoester)s, and the B-blocks arerelatively hydrophilic and comprise polyethylene glycol (PEG), saidcopolymers having a hydrophobic content of between 50.1 to 83% by weightand a hydrophilic content of between 17 to 49.9% by weight, and anoverall block copolymer molecular weight of between 2000 and 8000Daltons. The drug carriers exhibit water solubility at temperaturesbelow normal mammalian body temperatures and undergo reversible thermalgelation to then exist as a gel at temperatures equal to physiologicalmammalian body temperatures. The biodegradable, hydrophobic A polymerblock comprises a polyester or poly(ortho ester), in which the polyesteris synthesized from monomers selected from the group consisting ofD,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid,L-lactic acid, glycolide, glycolic acid, ε-caprolactone,ε-hydroxyhexanoic acid, γ-butyrolactone, γ-hydroxybutyric acid,δ-valerolactone, δ-hydroxyvaleric acid, hydroxybutyric acids, malicacid, and copolymers thereof and having an average molecular weight ofbetween about 600 and 3000 Daltons. The hydrophilic B-block segment ispreferably polyethylene glycol (PEG) having an average molecular weightof between about 500 and 2200 Daltons.

Additional biodegradable thermoplastic polyesters include AtriGel®(provided by Atrix Laboratories, Inc.) and/or those disclosed, e.g., inU.S. Pat. Nos. 5,324,519; 4,938,763; 5,702,716; 5,744,153; and5,990,194; wherein the suitable biodegradable thermoplastic polyester isdisclosed as a thermoplastic polymer. Examples of suitable biodegradablethermoplastic polyesters include polylactides, polyglycolides,polycaprolactones, copolymers thereof, terpolymers thereof, and anycombinations thereof. In some such embodiments, the suitablebiodegradable thermoplastic polyester is a polylactide, a polyglycolide,a copolymer thereof, a terpolymer thereof, or a combination thereof. Inone embodiment, the biodegradable thermoplastic polyester is 50/50poly(DL-lactide-co-glycolide) having a carboxy terminal group; ispresent in about 30 wt. % to about 40 wt. % of the composition; and hasan average molecular weight of about 23,000 to about 45,000.Alternatively, in another embodiment, the biodegradable thermoplasticpolyester is 75/25 poly (DL-lactide-co-glycolide) without a carboxyterminal group; is present in about 40 wt. % to about 50 wt. % of thecomposition; and has an average molecular weight of about 15,000 toabout 24,000. In further or alternative embodiments, the terminal groupsof the poly(DL-lactide-co-glycolide) are either hydroxyl, carboxyl, orester depending upon the method of polymerization. Polycondensation oflactic or glycolic acid provides a polymer with terminal hydroxyl andcarboxyl groups. Ring-opening polymerization of the cyclic lactide orglycolide monomers with water, lactic acid, or glycolic acid providespolymers with the same terminal groups. However, ring-opening of thecyclic monomers with a monofunctional alcohol such as methanol, ethanol,or 1-dodecanol provides a polymer with one hydroxyl group and one esterterminal groups. Ring-opening polymerization of the cyclic monomers witha diol such as 1,6-hexanediol or polyethylene glycol provides a polymerwith only hydroxyl terminal groups.

Since the polymer systems of thermoreversible gels dissolve morecompletely at reduced temperatures, methods of solubilization includeadding the required amount of polymer to the amount of water to be usedat reduced tempertures. Generally after wetting the polymer by shaking,the mixture is capped and placed in a cold chamber or in a thermostaticcontainer at about 0-10° C. in order to dissolve the polymer. Themixture is stirred or shaken to bring about a more rapid dissolution ofthe thermoreversible gel polymer. The cytotoxic agent and variousadditives such as buffers, salts, and preservatives are subsequentlyadded and dissolved. In some instances the cytotoxic agent and/or otherpharmaceutically active agent is suspended if it is insoluble in water.The pH is modulated by the addition of appropriate buffering agentsround window membrane mucoadhesive characteristics are optionallyimparted to a thermoreversible gel by incorporation of round windowmembrane mucoadhesive carbomers, such as Carbopol® 934P, to thecomposition (Majithiya et al, AAPS PharmSciTech (2006), 7(3), p. E1;EP0551626, both of which is incorporated herein by reference for suchdisclosure).

In one embodiment are auris-acceptable pharmaceutical gel formulationswhich do not require the use of an added viscosity enhancing agent. Suchgel formulations incorporate at least one pharmaceutically acceptablebuffer. In one aspect is a gel formulation comprising a cytotoxic agentand a pharmaceutically acceptable buffer. In another embodiment, thepharmaceutically acceptable excipient or carrier is a gelling agent.

In other embodiments, useful cytotoxic agent auris-acceptablepharmaceutical formulations also include one or more pH adjusting agentsor buffering agents to provide an endolymph or perilymph suitable pH.Suitable pH adjusting agents or buffers include, but are not limited toacetate, bicarbonate, ammonium chloride, citrate, phosphate,pharmaceutically acceptable salts thereof and combinations or mixturesthereof. Such pH adjusting agents and buffers are included in an amountrequired to maintain pH of the composition between a pH of about 5 andabout 9, in one embodiment a pH between about 6.5 to about 7.5, and inyet another embodiment at a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5. In one embodiment, when one or more buffers areutilized in the formulations of the present disclosure, they arecombined, e.g., with a pharmaceutically acceptable vehicle and arepresent in the final formulation, e.g., in an amount ranging from about0.1% to about 20%, from about 0.5% to about 10%. In certain embodimentsof the present disclosure, the amount of buffer included in the gelformulations are an amount such that the pH of the gel formulation doesnot interfere with the auris media or auris interna's natural bufferingsystem, or does not interfere with the natural pH of the endolymph orperilymph: depending on where in the cochlea the cytotoxic agentformulation is targeted. In some embodiments, from about 10 μM to about200 mM concentration of a buffer is present in the gel formulation. Incertain embodiments, from about a 5 mM to about a 200 mM concentrationof a buffer is present. In certain embodiments, from about a 20 mM toabout a 100 mM concentration of a buffer is present. In one embodimentis a buffer such as acetate or citrate at slightly acidic pH. In oneembodiment the buffer is a sodium acetate buffer having a pH of about4.5 to about 6.5. In one embodiment the buffer is a sodium citratebuffer having a pH of about 5.0 to about 8.0, or about 5.5 to about 7.0.

In an alternative embodiment, the buffer used istris(hydroxymethyl)aminomethane, bicarbonate, carbonate or phosphate atslightly basic pH. In one embodiment, the buffer is a sodium bicarbonatebuffer having a pH of about 6.5 to about 8.5, or about 7.0 to about 8.0.In another embodiment the buffer is a sodium phosphate dibasic bufferhaving a pH of about 6.0 to about 9.0.

Also described herein are controlled release formulations or devicescomprising a cytotoxic agent and a viscosity enhancing agent. Suitableviscosity-enhancing agents include by way of example only, gellingagents and suspending agents. In one embodiment, the enhanced viscosityformulation does not include a buffer. In other embodiments, theenhanced viscosity formulation includes a pharmaceutically acceptablebuffer. Sodium chloride or other tonicity agents are optionally used toadjust tonicity, if necessary.

By way of example only, the auris-acceptable viscosity agent includehydroxypropyl methylcellulose, hydroxyethyl cellulose,polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodiumchondroitin sulfate, sodium hyaluronate. Other viscosity enhancingagents compatible with the targeted auris structure include, but are notlimited to, acacia (gum arabic), agar, aluminum magnesium silicate,sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer,carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose(MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus,dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite,lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch,wheat starch, rice starch, potato starch, gelatin, sterculia gum,xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethylcellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethylcellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose,poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline,povidone, propylene carbonate, methyl vinyl ether/maleic anhydridecopolymer (PVM/MA), poly(methoxyethyl methacrylate),poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose,hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose(CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda®(dextrose, maltodextrin and sucralose) or combinations thereof. Inspecific embodiments, the viscosity-enhancing excipient is a combinationof MCC and CMC. In another embodiment, the viscosity-enhancing agent isa combination of carboxymethylated chitosan, or chitin, and alginate.The combination of chitin and alginate with the cytotoxic agentsdisclosed herein acts as a controlled release formulation, restrictingthe diffusion of the cytotoxic agents from the formulation. Moreover,the combination of carboxymethylated chitosan and alginate is optionallyused to assist in increasing the permeability of the cytotoxic agentsthrough the round window membrane.

In some embodiments is an enhanced viscosity formulation, comprisingfrom about 0.1 mM and about 100 mM of a cytotoxic agent, apharmaceutically acceptable viscosity agent, and water for injection,the concentration of the viscosity agent in the water being sufficientto provide a enhanced viscosity formulation with a final viscosity fromabout 100 to about 100,000 cP. In certain embodiments, the viscosity ofthe gel is in the range from about 100 to about 50,000 cP, about 100 cPto about 1,000 cP, about 500 cP to about 1500 cP, about 1000 cP to about3000 cP, about 2000 cP to about 8,000 cP, about 4,000 cP to about 50,000cP, about 10,000 cP to about 500,000 cP, about 15,000 cP to about1,000,000 cP. In other embodiments, when an even more viscous medium isdesired, the biocompatible gel comprises at least about 35%, at leastabout 45%, at least about 55%, at least about 65%, at least about 70%,at least about 75%, or even at least about 80% or so by weight of thecytotoxic agent. In highly concentrated samples, the biocompatibleenhanced viscosity formulation comprises at least about 25%, at leastabout 35%, at least about 45%, at least about 55%, at least about 65%,at least about 75%, at least about 85%, at least about 90% or at leastabout 95% or more by weight of the cytotoxic agent.

In some embodiments, the viscosity of the gel formulations presentedherein are measured by any means described. For example, in someembodiments, an LVDV-II+CP Cone Plate Viscometer and a Cone SpindleCPE-40 is used to calculate the viscosity of the gel formulationdescribed herein. In other embodiments, a Brookfield (spindle and cup)viscometer is used to calculate the viscosity of the gel formulationdescribed herein. In some embodiments, the viscosity ranges referred toherein are measured at room temperature. In other embodiments, theviscosity ranges referred to herein are measured at body temperature(e.g., at the average body temperature of a healthy human).

In one embodiment, the pharmaceutically acceptable enhanced viscosityauris-acceptable formulation comprises at least one cytotoxic agent andat least one gelling agent. Suitable gelling agents for use inpreparation of the gel formulation include, but are not limited to,celluloses, cellulose derivatives, cellulose ethers (e.g.,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose,hydroxypropylcellulose, methylcellulose), guar gum, xanthan gum, locustbean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth,carboxyvinyl polymers, carrageenan, paraffin, petrolatum and anycombinations or mixtures thereof. In some other embodiments,hydroxypropylmethylcellulose (Methocel®) is utilized as the gellingagent. In certain embodiments, the viscosity enhancing agents describedherein are also utilized as the gelling agent for the gel formulationspresented herein.

In some embodiments, the otic therapeutic agents disclosed herein aredispensed as an auris-acceptable paint. As used herein, paints (alsoknown as film formers) are solutions comprised of a solvent, a monomeror polymer, an active agent, and optionally one or morepharmaceutically-acceptable excipients. After application to a tissue,the solvent evaporates leaving behind a thin coating comprised of themonomers or polymers, and the active agent. The coating protects activeagents and maintains them in an immobilized state at the site ofapplication. This decreases the amount of active agent which may be lostand correspondingly increases the amount delivered to the subject. Byway of non-limiting example, paints include collodions (e.g. FlexibleCollodion, USP), and solutions comprising saccharide siloxane copolymersand a cross-linking agent. Collodions are ethyl ether/ethanol solutionscontaining pyroxylin (a nitrocellulose). After application, the ethylether/ethanol solution evaporates leaving behind a thin film ofpyroxylin. In solutions comprising saccharide siloxane copolymers, thesaccharide siloxane copolymers form the coating after evaporation of thesolvent initiates the cross-linking of the saccharide siloxanecopolymers. For additional disclosures regarding paints, see Remington:The Science and Practice of Pharmacy which is hereby incorporated withrespect to this subject matter. The paints contemplated for use herein,are flexible such that they do not interfere with the propagation ofpressure waves through the ear. Further, the paints may be applied as aliquid (i.e. solution, suspension, or emulsion), a semisolid (i.e. agel, foam, paste, or jelly) or an aerosol.

In some embodiments, the otic therapeutic agents disclosed herein aredispensed as a controlled-release foam. Examples of suitable foamablecarriers for use in the compositions disclosed herein include, but arenot limited to, alginate and derivatives thereof, carboxymethylcelluloseand derivatives thereof, collagen, polysaccharides, including, forexample, dextran, dextran derivatives, pectin, starch, modified starchessuch as starches having additional carboxyl and/or carboxamide groupsand/or having hydrophilic side-chains, cellulose and derivativesthereof, agar and derivatives thereof, such as agar stabilised withpolyacrylamide, polyethylene oxides, glycol methacrylates, gelatin, gumssuch as xanthum, guar, karaya, gellan, arabic, tragacanth and locustbean gum, or combinations thereof. Also suitable are the salts of theaforementioned carriers, for example, sodium alginate. The formulationoptionally further comprises a foaming agent, which promotes theformation of the foam, including a surfactant or external propellant.Examples of suitable foaming agents include cetrimide, lecithin, soaps,silicones and the like. Commercially available surfactants such asTween® are also suitable.

In some embodiments, other gel formulations are useful depending uponthe particular cytotoxic agent, other pharmaceutical agent orexcipients/additives used, and as such are considered to fall within thescope of the present disclosure. For example, othercommercially-available glycerin-based gels, glycerin-derived compounds,conjugated, or crosslinked gels, matrices, hydrogels, and polymers, aswell as gelatins and their derivatives, alginates, and alginate-basedgels, and even various native and synthetic hydrogel andhydrogel-derived compounds are all expected to be useful in thecytotoxic agent formulations described herein. In some embodiments,auris-acceptable gels include, but are not limited to, alginatehydrogels SAF®-Gel (ConvaTec, Princeton, N.J.), Duoderm® Hydroactive Gel(ConvaTec), Nu-gel® (Johnson & Johnson Medical, Arlington, Tex.);Carrasyn® (V) Acemannan Hydrogel (Carrington Laboratories, Inc., Irving,Tex.); glycerin gels Elta® Hydrogel (Swiss-American Products, Inc.,Dallas, Tex.) and K-Y® Sterile (Johnson & Johnson). In furtherembodiments, biodegradable biocompatible gels also represent compoundspresent in auris-acceptable formulations disclosed and described herein.

In some formulations developed for administration to a mammal, and forcompositions formulated for human administration, the auris-acceptablegel comprises substantially all of the weight of the composition. Inother embodiments, the auris-acceptable gel comprises as much as about98% or about 99% of the composition by weight. This is desirous when asubstantially non-fluid, or substantially viscous formulation is needed.In a further embodiment, when slightly less viscous, or slightly morefluid auris-acceptable pharmaceutical gel formulations are desired, thebiocompatible gel portion of the formulation comprises at least about50% by weight, at least about 60% by weight, at least about 70% byweight, or even at least about 80% or 90% by weight of the compound. Allintermediate integers within these ranges are contemplated to fallwithin the scope of this disclosure, and in some alternativeembodiments, even more fluid (and consequently less viscous)auris-acceptable gel compositions are formulated, such as for example,those in which the gel or matrix component of the mixture comprises notmore than about 50% by weight, not more than about 40% by weight, notmore than about 30% by weight, or even those than comprise not more thanabout 15% or about 20% by weight of the composition.

Auris-Acceptable Suspending Agents

In one embodiment, at least one cytotoxic agent is included in apharmaceutically acceptable enhanced viscosity formulation wherein theformulation further comprises at least one suspending agent, wherein thesuspending agent assists in imparting controlled release characteristicsto the formulation. In some embodiments, suspending agents also serve toincrease the viscosity of the auris-acceptable cytotoxic agentformulations and compositions.

Suspending agents include, by way of example only, compounds such aspolyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, orpolyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer(S630), sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose (hypromellose), hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like. In some embodiments, useful aqueous suspensions also containone or more polymers as suspending agents. Useful polymers includewater-soluble polymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers.

In one embodiment, the present disclosure provides auris-acceptable gelcompositions comprising a therapeutically effective amount of acytotoxic agent in a hydroxyethyl cellulose gel. Hydroxyethyl cellulose(HEC) is obtained as a dry powder which is reconstituted in water or anaqueous buffer solution to give the desired viscosity (generally about200 cps to about 30,000 cps, corresponding to about 0.2 to about 10%HEC). In one embodiment the concentration of HEC is between about 1% andabout 15%, about 1% and about 2%, or about 1.5% to about 2%.

In other embodiments, the auris-acceptable formulations, including gelformulations and viscosity-enhanced formulations, further includeexcipients, other medicinal or pharmaceutical agents, carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts, solubilizers, an antifoaming agent,an antioxidant, a dispersing agent, a wetting agent, a surfactant, andcombinations thereof.

Auris-Acceptable Actinic Radiation Curable Gel

In other embodiments, the gel is an actinic radiation curable gel, suchthat following administration to or near the targeted auris structure,use of actinic radiation (or light, including UV light, visible light,or infrared light) the desired gel properties are formed. By way ofexample only, fiber optics are used to provide the actinic radiation soas to form the desired gel properties. In some embodiments, the fiberoptics and the gel administration device form a single unit. In otherembodiments, the fiber optics and the gel administration device areprovided separately.

Auris-Acceptable Solvent Release Gel

In some embodiments, the gel is a solvent release gel such that thedesired gel properties are formed after administration to or near thetargeted auris structure, that is, as the solvent in the injected gelformulation diffuses out the gel, a gel having the desired gelproperties is formed. For example, a formulation that comprises sucroseacetate isobutyrate, a pharmaceutically acceptable solvent, one or moreadditives, and the cytotoxic agent is administered at or near the roundwindow membrane: diffusion of the solvent out of the injectedformulation provides a depot having the desired gel properties. Forexample, use of a water soluble solvent provides a high viscosity depotwhen the solvent diffuses rapidly out of the injected formulation. Onthe other hand, use of a hydrophobic solvent (e.g., benzyl benzoate)provides a less viscous depot. One example of an auris-acceptablesolvent release gel formulation is the SABER™ Delivery System marketedby DURECT Corporation.

Auris-Acceptable In Situ Forming Spongy Material

Also contemplated within the scope of the embodiments is the use of aspongy material, formed in situ in the auris interna or auris media. Insome embodiments, the spongy material is formed from hyaluronic acid orits derivatives. The spongy material is impregnated with a desiredcytotoxic agent and placed within the auris media so as to providecontrolled release of the cytotoxic agent within the auris media, or incontact with the round window membrane so as to provide controlledrelease of the cytotoxic agent into the auris interna. In someembodiments, the spongy material is biodegradable.

Round Window Membrane Mucoadhesives

Also contemplated within the scope of the embodiments is the addition ofa round window membrane mucoadhesive with the cytotoxic agentformulations and compositions and devices disclosed herein. The term‘mucoadhesion’ is used for materials that bind to the mucin layer of abiological membrane, such as the external membrane of the 3-layeredround window membrane. To serve as round window membrane mucoadhesivepolymers, the polymers possess some general physiochemical features suchas predominantly anionic hydrophilicity with numerous hydrogen bondforming groups, suitable surface property for wetting mucus/mucosaltissue surfaces or sufficient flexibility to penetrate the mucusnetwork.

Round window membrane mucoadhesive agents that are used with theauris-acceptable formulations include, but are not limited to, at leastone soluble polyvinylpyrrolidone polymer (PVP); a water-swellable, butwater-insoluble, fibrous, cross-linked carboxy-functional polymer; acrosslinked poly(acrylic acid) (e.g. Carbopol® 947P); a carbomerhomopolymer; a carbomer copolymer; a hydrophilic polysaccharide gum,maltodextrin, a cross-linked alignate gum gel, a water-dispersiblepolycarboxylated vinyl polymer, at least two particulate componentsselected from the group consisting of titanium dioxide, silicon dioxide,and clay, or a mixture thereof. The round window membrane mucoadhesiveagent is optionally used in combination with an auris-acceptableviscosity increasing excipient, or used alone to increase theinteraction of the composition with the mucosal layer target oticcomponent. In one non-limiting example, the mucoadhesive agent ismaltodextrin. In some embodiments, the mucoadhesive agent is an alginategum. When used, the round window membrane mucoadhesive characterimparted to the composition is at a level that is sufficient to deliveran effective amount of the cytotoxic agent composition to, for example,the mucosal layer of round window membrane or the crista fenestraecochleae in an amount that coats the mucosal membrane, and thereafterdeliver the composition to the affected areas, including by way ofexample only, the vestibular and/or cochlear structures of the aurisinterna. When used, the mucoadhesive characteristics of the compositionsprovided herein are determined, and using this information (along withthe other teachings provided herein), the appropriate amounts aredetermined. One method for determining sufficient mucoadhesivenessincludes monitoring changes in the interaction of the composition with amucosal layer, including but not limited to measuring changes inresidence or retention time of the composition in the absence andpresence of the mucoadhesive excipient.

Mucoadhesive agents have been described, for example, in U.S. Pat. Nos.6,638,521, 6,562,363, 6,509,028, 6,348,502, 6,319,513, 6,306,789,5,814,330, and 4,900,552, each of which is hereby incorporated byreference for such disclosure.

In another non-limiting example, a mucoadhesive agent is, for example,at least two particulate components selected from titanium dioxide,silicon dioxide, and clay, wherein the composition is not furtherdiluted with any liquid prior to administration and the level of silicondioxide, if present, is from about 3% to about 15%, by weight of thecomposition. Silicon dioxide, if present, includes fumed silicondioxide, precipitated silicon dioxide, coacervated silicon dioxide, gelsilicon dioxide, and mixtures thereof. Clay, if present, includes kaolinminerals, serpentine minerals, smectites, illite or a mixture thereof.For example, clay includes laponite, bentonite, hectorite, saponite,montmorillonites or a mixture thereof.

In one non-limiting example, the round window membrane mucoadhesiveagent is maltodextrin. Maltodextrin is a carbohydrate produced by thehydrolysis of starch that is optionally derived from corn, potato, wheator other plant products. Maltodextrin is optionally used either alone orin combination with other round window membrane mucoadhesive agents toimpart mucoadhesive characteristics on the compositions disclosedherein. In one embodiment, a combination of maltodextrin and a carbopolpolymer are used to increase the round window membrane mucoadhesivecharacteristics of the compositions or devices disclosed herein.

In another embodiment, the round window membrane mucoadhesive agent isan alkyl-glycoside and/or a saccharide alkyl ester. As used herein, an“alkyl-glycoside” means a compound comprising any hydrophilic saccharide(e.g. sucrose, maltose, or glucose) linked to a hydrophobic alkyl. Insome embodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl-glycoside comprises a sugar linked toa hydrophobic alkyl (e.g., an alkyl comprising about 6 to about 25carbon atoms) by an amide linkage, an amine linkage, a carbamatelinkage, an ether linkage, a thioether linkage, an ester linkage, athioester linkage, a glycosidic linkage, a thioglycosidic linkage,and/or a ureide linkage. In some embodiments, the round window membranemucoadhesive agent is a hexyl-, heptyl-, octyl-, nonyl-, decyl-,undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-, hexadecyl-,heptadecyl-, and octadecyl α- or β-D-maltoside; hexyl-, heptyl-, octyl-,nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-,hexadecyl-, heptadecyl-, and octadecyl α- or β-D-glucoside; hexyl-,heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-,tetradecyl, pentadecyl-, hexadecyl-, heptadecyl-, and octadecyl α- orβ-D-sucroside; hexyl-, heptyl-, octyl-, dodecyl-, tridecyl-, andtetradecyl-β-D-thiomaltoside; heptyl- or octyl-1-thio-α- orβ-D-glucopyranoside; alkyl thiosucroses; alkyl maltotriosides; longchain aliphatic carbonic acid amides of sucrose α-amino-alkyl ethers;derivatives of palatinose or isomaltamine linked by an amide linkage toan alkyl chain and derivatives of isomaltamine linked by urea to analkyl chain; long chain aliphatic carbonic acid ureides of sucroseβ-amino-alkyl ethers and long chain aliphatic carbonic acid amides ofsucrose β-amino-alkyl ethers. In some embodiments, the round windowmembrane mucoadhesive agent is an alkyl-glycoside wherein the alkylglycoside is maltose, sucrose, glucose, or a combination thereof linkedby a glycosidic linkage to an alkyl chain of 9-16 carbon atoms (e.g.,nonyl-, decyl-, dodecyl- and tetradecyl sucroside; nonyl-, decyl-,dodecyl- and tetradecyl glucoside; and nonyl-, decyl-, dodecyl- andtetradecyl maltoside). In some embodiments, the round window membranemucoadhesive agent is an alkyl-glycoside wherein the alkyl glycoside isdodecylmaltoside, tridecylmaltoside, and tetradecylmaltoside.

In some embodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl-glycoside is a disaccharide with atleast one glucose. In some embodiments, the auris acceptable penetrationenhancer is a surfactant comprisingα-D-glucopyranosyl-β-glycopyranoside,n-Dodecyl-4-O-α-D-glucopyranosyl-β-glycopyranoside, and/orn-tetradecyl-4-O-α-D-glucopyranosyl-β-glycopyranoside. In someembodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl-glycoside has a critical miscelleconcentration (CMC) of less than about 1 mM in pure water or in aqueoussolutions. In some embodiments, the round window membrane mucoadhesiveagent is an alkyl-glycoside wherein an oxygen atom within thealkyl-glycoside is substituted with a sulfur atom. In some embodiments,the round window membrane mucoadhesive agent is an alkyl-glycosidewherein the alkylglycoside is the β anomer. In some embodiments, theround window membrane mucoadhesive agent is an alkyl-glycoside whereinthe alkylglycoside comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.5%, or 99.9% of the β anomer.

Auris-Acceptable Controlled Release Particles

Cytotoxic agents and/or other pharmaceutical agents disclosed herein areoptionally incorporated within controlled release particles, lipidcomplexes, liposomes, nanoparticles, microparticles, microspheres,coacervates, nanocapsules or other agents which enhance or facilitatethe localized delivery of the cytotoxic agent. In some embodiments, asingle enhanced viscosity formulation is used, in which at least onecytotoxic agent is present, while in other embodiments, a pharmaceuticalformulation that comprises a mixture of two or more distinct enhancedviscosity formulations is used, in which at least one cytotoxic agent ispresent. In some embodiments, combinations of sols, gels and/orbiocompatible matrices is also employed to provide desirablecharacteristics of the controlled release cytotoxic agent compositionsor formulations. In certain embodiments, the controlled releasecytotoxic agent formulations or compositions are cross-linked by one ormore agents to alter or improve the properties of the composition.

Examples of microspheres relevant to the pharmaceutical formulationsdisclosed herein include: Luzzi, L. A., J. Pharm. Psy. 59:1367 (1970);U.S. Pat. No. 4,530,840; Lewis, D. H., “Controlled Release of BioactiveAgents from Lactides/Glycolide Polymers” in Biodegradable Polymers asDrug Delivery Systems, Chasin, M. and Langer, R., eds., Marcel Decker(1990); U.S. Pat. No. 4,675,189; Beck et al., “Poly(lactic acid) andPoly(lactic acid-co-glycolic acid) Contraceptive Delivery Systems,” inLong Acting Steroid Contraception, Mishell, D. R., ed., Raven Press(1983); U.S. Pat. No. 4,758,435; U.S. Pat. No. 3,773,919; U.S. Pat. No.4,474,572. Examples of protein therapeutics formulated as microspheresinclude: U.S. Pat. No. 6,458,387; U.S. Pat. No. 6,268,053; U.S. Pat. No.6,090,925; U.S. Pat. No. 5,981,719; and U.S. Pat. No. 5,578,709, and areherein incorporated by reference for such disclosure.

Microspheres usually have a spherical shape, although irregularly-shapedmicroparticles are possible. Microspheres may vary in size, ranging fromsubmicron to 1000 micron diameters. Microspheres suitable for use withthe auris-acceptable formulations disclosed herein are submicron to 250micron diameter microspheres, allowing administration by injection witha standard gauge needle. The auris-acceptable microspheres are preparedby any method which produces microspheres in a size range acceptable foruse in an injectable composition. Injection is optionally accomplishedwith standard gauge needles used for administering liquid compositions.

Suitable examples of polymeric matrix materials for use in theauris-acceptable controlled release particles herein includepoly(glycolic acid), poly-d,l-lactic acid, poly-l-lactic acid,copolymers of the foregoing, poly(aliphatic carboxylic acids),copolyoxalates, polycaprolactone, polydioxonene, poly(orthocarbonates),poly(acetals), poly(lactic acid-caprolactone), polyorthoesters,poly(glycolic acid-caprolactone), polydioxonene, polyanhydrides,polyphosphazines, and natural polymers including albumin, casein, andsome waxes, such as, glycerol mono- and distearate, and the like.Various commercially available poly (lactide-co-glycolide) materials(PLGA) are optionally used in the method disclosed herein. For example,poly (d,l-lactic-co-glycolic acid) is commercially available fromBoehringer-Ingelheim as RESOMER RG 503H. This product has a mole percentcomposition of 50% lactide and 50% glycolide. These copolymers areavailable in a wide range of molecular weights and ratios of lactic acidto glycolic acid. One embodiment includes the use of the polymerpoly(d,l-lactide-co-glycolide). The molar ratio of lactide to glycolidein such a copolymer includes the range of from about 95:5 to about50:50.

The molecular weight of the polymeric matrix material is of someimportance. The molecular weight should be high enough so that it formssatisfactory polymer coatings, i.e., the polymer should be a good filmformer. Usually, a satisfactory molecular weight is in the range of5,000 to 500,000 daltons. The molecular weight of a polymer is alsoimportant from the point of view that molecular weight influences thebiodegradation rate of the polymer. For a diffusional mechanism of drugrelease, the polymer should remain intact until all of the drug isreleased from the microparticles and then degrade. The drug is alsoreleased from the microparticles as the polymeric excipient bioerodes.By an appropriate selection of polymeric materials a microsphereformulation is made such that the resulting microspheres exhibit bothdiffusional release and biodegradation release properties. This isuseful in affording multiphasic release patterns.

A variety of methods are known by which compounds are encapsulated inmicrospheres. In these methods, the cytotoxic agent is generallydispersed or emulsified, using stirrers, agitators, or other dynamicmixing techniques, in a solvent containing a wall-forming material.Solvent is then removed from the microspheres, and thereafter themicrosphere product is obtained.

In one embodiment, controlled release cytotoxic agent formulations aremade through the incorporation of the cytotoxic agents and/or otherpharmaceutical agents into ethylene-vinyl acetate copolymer matrices.(See U.S. Pat. No. 6,083,534, incorporated herein for such disclosure).In another embodiment, cytotoxic agents are incorporated into poly(lactic-glycolic acid) or poly-L-lactic acid microspheres. Id. In yetanother embodiment, the cytotoxic agents are encapsulated into alginatemicrospheres. (See U.S. Pat. No. 6,036,978, incorporated herein for suchdisclosure). Biocompatible methacrylate-based polymers to encapsulatethe cytotoxic agent compounds or compositions are optionally used in theformulations and methods disclosed herein. A wide range ofmethacrylate-based polymer systems are commercially available, such asthe EUDRAGIT polymers marketed by Evonik. One useful aspect ofmethacrylate polymers is that the properties of the formulation arevaried by incorporating various co-polymers. For example, poly(acrylicacid-co-methylmethacrylate) microparticles exhibit enhanced mucoadhesionproperties as the carboxylic acid groups in the poly(acrylic acid) formhydrogen bonds with mucin (Park et al, Pharm. Res. (1987) 4(6):457-464).Variation of the ratio between acrylic acid and methylmethacrylatemonomers serves to modulate the properties of the co-polymer.Methacrylate-based microparticles have also been used in proteintherapeutic formulations (Naha et al, Journal of Microencapsulation 4Feb., 2008 (online publication)). In one embodiment, the enhancedviscosity auris-acceptable formulations described herein comprisescytotoxic agent microspheres wherein the microspheres are formed from amethacrylate polymer or copolymer. In an additional embodiment, theenhanced viscosity formulation described herein comprises cytotoxicagent microspheres wherein the microspheres are mucoadhesive. Othercontrolled release systems, including incorporation or deposit ofpolymeric materials or matrices onto solid or hollow spheres containingcytotoxic agents, are also explicitly contemplated within theembodiments disclosed herein. The types of controlled release systemsavailable without significantly losing activity of the cytotoxic agentare determined using the teachings, examples, and principles disclosedherein

An example of a conventional microencapsulation process forpharmaceutical preparations is shown in U.S. Pat. No. 3,737,337,incorporated herein by reference for such disclosure. The cytotoxicagent substances to be encapsulated or embedded are dissolved ordispersed in the organic solution of the polymer (phase A), usingconventional mixers, including (in the preparation of dispersion)vibrators and high-speed stirrers, etc. The dispersion of phase (A),containing the core material in solution or in suspension, is carriedout in the aqueous phase (B), again using conventional mixers, such ashigh-speed mixers, vibration mixers, or even spray nozzles, in whichcase the particle size of the microspheres will be determined not onlyby the concentration of phase (A), but also by the emulsate ormicrosphere size. With conventional techniques for themicroencapsulation of cytotoxic agents, the microspheres form when thesolvent containing an active agent and a polymer is emulsified ordispersed in an immiscible solution by stirring, agitating, vibrating,or some other dynamic mixing technique, often for a relatively longperiod of time.

Methods for the construction of microspheres are also described in U.S.Pat. No. 4,389,330, and U.S. Pat. No. 4,530,840, incorporated herein byreference for such disclosure. The desired cytotoxic agent is dissolvedor dispersed in an appropriate solvent. To the agent-containing mediumis added the polymeric matrix material in an amount relative to theactive ingredient which gives a product of the desired loading of activeagent. Optionally, all of the ingredients of the cytotoxic agentmicrosphere product can be blended in the solvent medium together.Suitable solvents for the agent and the polymeric matrix materialinclude organic solvents such as acetone, halogenated hydrocarbons suchas chloroform, methylene chloride and the like, aromatic hydrocarboncompounds, halogenated aromatic hydrocarbon compounds, cyclic ethers,alcohols, ethyl acetate and the like.

The mixture of ingredients in the solvent is emulsified in acontinuous-phase processing medium; the continuous-phase medium beingsuch that a dispersion of microdroplets containing the indicatedingredients is formed in the continuous-phase medium. Naturally, thecontinuous-phase processing medium and the organic solvent must beimmiscible, and includes water although nonaqueous media such as xyleneand toluene and synthetic oils and natural oils are optionally used.Optionally, a surfactant is added to the continuous-phase processingmedium to prevent the microparticles from agglomerating and to controlthe size of the solvent microdroplets in the emulsion. A preferredsurfactant-dispersing medium combination is a 1 to 10 wt. % poly (vinylalcohol) in water mixture. The dispersion is formed by mechanicalagitation of the mixed materials. An emulsion is optionally formed byadding small drops of the active agent-wall forming material solution tothe continuous phase processing medium. The temperature during theformation of the emulsion is not especially critical but influences thesize and quality of the microspheres and the solubility of the drug inthe continuous phase. It is desirable to have as little of the agent inthe continuous phase as possible. Moreover, depending on the solvent andcontinuous-phase processing medium employed, the temperature must not betoo low or the solvent and processing medium will solidify or theprocessing medium will become too viscous for practical purposes, or toohigh that the processing medium will evaporate, or that the liquidprocessing medium will not be maintained. Moreover, the temperature ofthe medium cannot be so high that the stability of the particular agentbeing incorporated in the microspheres is adversely affected.Accordingly, the dispersion process is conducted at any temperaturewhich maintains stable operating conditions, which preferred temperaturebeing about 15° C. to 60° C., depending upon the drug and excipientselected.

The dispersion which is formed is a stable emulsion and from thisdispersion the organic solvent immiscible fluid is optionally partiallyremoved in the first step of the solvent removal process. The solvent isremoved by techniques such as heating, the application of a reducedpressure or a combination of both. The temperature employed to evaporatesolvent from the microdroplets is not critical, but should not be thathigh that it degrades the cytotoxic agent employed in the preparation ofa given microparticle, nor should it be so high as to evaporate solventat such a rapid rate to cause defects in the wall forming material.Generally, from 5 to 75%, of the solvent is removed in the first solventremoval step.

After the first stage, the dispersed microparticles in the solventimmiscible fluid medium are isolated from the fluid medium by anyconvenient means of separation. Thus, for example, the fluid is decantedfrom the microsphere or the microsphere suspension is filtered. Stillother, various combinations of separation techniques are used ifdesired.

Following the isolation of the microspheres from the continuous-phaseprocessing medium, the remainder of the solvent in the microspheres isremoved by extraction. In this step, the microspheres are suspended inthe same continuous-phase processing medium used in step one, with orwithout surfactant, or in another liquid. The extraction medium removesthe solvent from the microspheres and yet does not dissolve themicrospheres. During the extraction, the extraction medium withdissolved solvent is optionally removed and replaced with freshextraction medium. This is best done on a continual basis. The rate ofextraction medium replenishment of a given process is a variable whichis determined at the time the process is performed and, therefore, noprecise limits for the rate must be predetermined. After the majority ofthe solvent has been removed from the microspheres, the microspheres aredried by exposure to air or by other conventional drying techniques suchas vacuum drying, drying over a desiccant, or the like. This process isvery efficient in encapsulating the cytotoxic agent since core loadingsof up to 80 wt. %, preferably up to 60 wt. % are obtained.

Alternatively, controlled release microspheres containing a cytotoxicagent is prepared through the use of static mixers. Static or motionlessmixers consist of a conduit or tube in which is received a number ofstatic mixing agents. Static mixers provide homogeneous mixing in arelatively short length of conduit, and in a relatively short period oftime. With static mixers, the fluid moves through the mixer, rather thansome part of the mixer, such as a blade, moving through the fluid.

A static mixer is optionally used to create an emulsion. When using astatic mixer to form an emulsion, several factors determine emulsionparticle size, including the density and viscosity of the varioussolutions or phases to be mixed, volume ratio of the phases, interfacialtension between the phases, static mixer parameters (conduit diameter;length of mixing element; number of mixing elements), and linearvelocity through the static mixer. Temperature is a variable because itaffects density, viscosity, and interfacial tension. The controllingvariables are linear velocity, sheer rate, and pressure drop per unitlength of static mixer.

In order to create microspheres containing a cytotoxic agent using astatic mixer process, an organic phase and an aqueous phase arecombined. The organic and aqueous phases are largely or substantiallyimmiscible, with the aqueous phase constituting the continuous phase ofthe emulsion. The organic phase includes a cytotoxic agent as well as awall-forming polymer or polymeric matrix material. The organic phase isprepared by dissolving a cytotoxic agent in an organic or other suitablesolvent, or by forming a dispersion or an emulsion containing thecytotoxic agent. The organic phase and the aqueous phase are pumped sothat the two phases flow simultaneously through a static mixer, therebyforming an emulsion which comprises microspheres containing thecytotoxic agent encapsulated in the polymeric matrix material. Theorganic and aqueous phases are pumped through the static mixer into alarge volume of quench liquid to extract or remove the organic solvent.Organic solvent is optionally removed from the microspheres while theyare washing or being stirred in the quench liquid. After themicrospheres are washed in a quench liquid, they are isolated, asthrough a sieve, and dried.

In one embodiment, microspheres are prepared using a static mixer. Theprocess is not limited to the solvent extraction technique discussedabove, but is used with other encapsulation techniques. For example, theprocess is optionally used with a phase separation encapsulationtechnique. To do so, an organic phase is prepared that comprises acytotoxic agent suspended or dispersed in a polymer solution. Thenon-solvent second phase is free from solvents for the polymer andactive agent. A preferred non-solvent second phase is silicone oil. Theorganic phase and the non-solvent phase are pumped through a staticmixer into a non-solvent quench liquid, such as heptane. The semi-solidparticles are quenched for complete hardening and washing. The processof microencapsulation includes spray drying, solvent evaporation, acombination of evaporation and extraction, and melt extrusion.

In another embodiment, the microencapsulation process involves the useof a static mixer with a single solvent. This process is described indetail in U.S. application Ser. No. 08/338,805, herein incorporated byreference for such disclosure. An alternative process involves the useof a static mixer with co-solvents. In this process, biodegradablemicrospheres comprising a biodegradable polymeric binder and a cytotoxicagent are prepared, which comprises a blend of at least twosubstantially non-toxic solvents, free of halogenated hydrocarbons todissolve both the agent and the polymer. The solvent blend containingthe dissolved agent and polymer is dispersed in an aqueous solution toform droplets. The resulting emulsion is then added to an aqueousextraction medium preferably containing at least one of the solvents ofthe blend, whereby the rate of extraction of each solvent is controlled,whereupon the biodegradable microspheres containing the pharmaceuticallyactive agent are formed. This process has the advantage that lessextraction medium is required because the solubility of one solvent inwater is substantially independent of the other and solvent selection isincreased, especially with solvents that are particularly difficult toextract.

Nanoparticles are also contemplated for use with the cytotoxic agentsdisclosed herein. Nanoparticles are material structures of about 100 nmor less in size. One use of nanoparticles in pharmaceutical formulationsis the formation of suspensions as the interaction of the particlesurface with solvent is strong enough to overcome differences indensity. Nanoparticle suspensions are sterilized as the nanoparticlesare small enough to be subjected to sterilizing filtration (see, e.g.,U.S. Pat. No. 6,139,870, herein incorporated by reference for suchdisclosure). Nanoparticles comprise at least one hydrophobic,water-insoluble and water-indispersible polymer or copolymer emulsifiedin a solution or aqueous dispersion of surfactants, phospholipids orfatty acids. The cytotoxic agent is optionally introduced with thepolymer or the copolymer into the nanoparticles.

Lipid nanocapsules as controlled release structures, as well forpenetrating the round window membrane and reaching auris interna and/orauris media targets, is also contemplated herein. Lipid nanocapsules areoptionally formed by emulsifying capric and caprylic acid triglycerides(Labrafac WL 1349; avg. mw 512), soybean lecithin (LIPOID® S75-3; 69%phosphatidylcholine and other phospholipids), surfactant (for example,Solutol HS15), a mixture of polyethylene glycol 660 hydroxystearate andfree polyethylene glycol 660; NaCl and water. The mixture is stirred atroom temperature to obtain an oil emulsion in water. After progressiveheating at a rate of 4° C./min under magnetic stirring, a short intervalof transparency should occur close to 70° C., and the inverted phase(water droplets in oil) obtained at 85° C. Three cycles of cooling andheating is then applied between 85° C. and 60° C. at the rate of 4°C./min, and a fast dilution in cold water at a temperature close to 0°C. to produce a suspension of nanocapsules. To encapsulate the cytotoxicagents, the agent is optionally added just prior to the dilution withcold water.

Cytotoxic agents are also inserted into the lipid nanocapsules byincubation for 90 minutes with an aqueous micellar solution of the aurisactive agent. The suspension is then vortexed every 15 minutes, and thenquenched in an ice bath for 1 minute.

Suitable auris-acceptable surfactants are, by way of example, cholicacid or taurocholic acid salts. Taurocholic acid, the conjugate formedfrom cholic acid and taurine, is a fully metabolizable sulfonic acidsurfactant. An analog of taurocholic acid, tauroursodeoxycholic acid(TUDCA), is a naturally occurring bile acid and is a conjugate oftaurine and ursodeoxycholic acid (UDCA). Other naturally occurringanionic (e.g., galactocerebroside sulfate), neutral (e.g.,lactosylceramide) or zwitterionic surfactants (e.g., sphingomyelin,phosphatidyl choline, palmitoyl camitine) are optionally used to preparenanoparticles.

The auris-acceptable phospholipids are chosen, by way of example, fromnatural, synthetic or semi-synthetic phospholipids; lecithins(phosphatidylcholine) such as, for example, purified egg or soyalecithins (lecithin E100, lecithin E80 and phospholipons, for examplephospholipon 90), phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol,dipalmitoylphosphatidylcholine, dipalmitoylglycerophosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine andphosphatidic acid or mixtures thereof are used more particularly.

Fatty acids for use with the auris-acceptable formulations are chosenfrom, by way of example, lauric acid, mysristic acid, palmitic acid,stearic acid, isostearic acid, arachidic acid, behenic acid, oleic acid,myristoleic acid, palmitoleic acid, linoleic acid, alpha-linoleic acid,arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoicacid, and the like.

Suitable auris-acceptable surfactants are selected from known organicand inorganic pharmaceutical excipients. Such excipients include variouspolymers, low molecular weight oligomers, natural products, andsurfactants. Preferred surface modifiers include nonionic and ionicsurfactants. Two or more surface modifiers are used in combination.

Representative examples of auris-acceptable surfactants include cetylpyridinium chloride, gelatin, casein, lecithin (phosphatides), dextran,glycerol, gum acacia, cholesterol, tragacanth, stearic acid, calciumstearate, glycerol monostearate, cetostearyl alcohol, cetomacrogolemulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fattyacid esters; dodecyl trimethyl ammonium bromide,polyoxyethylenestearates, colloidal silicon dioxide, phosphates, sodiumdodecylsulfate, carboxymethylcellulose calcium, hydroxypropyl cellulose(HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (HPMC),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethyl-cellulose phthalate,noncrystalline cellulose, magnesium aluminum silicate, triethanolamine,polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP),4-(1,1,3,3-tetaamethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol, superione, and triton),poloxamers, poloxamines, a charged phospholipid such as dimyristoylphophatidyl glycerol, dioctylsulfosuccinate (DOSS); Tetronic® 1508,dialkylesters of sodium sulfosuccinic acid, Duponol P, Tritons X-200,Crodestas F-110, p-isononylphenoxypoly-(glycidol), Crodestas SL-40(Croda, Inc.); and SA9OHCO, which is C₁₈H₃₇CH₂ (CON(CH₃)—CH₂ (CHOH)₄(CH₂OH)₂ (Eastman Kodak Co.); decanoyl-N-methylglucamide; n-decylβ-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecylβ-D-glucopyranoside; n-dodecyl β-D-maltoside;heptanoyl-N-methylglucamide; n-heptyl-β-D-glucopyranoside; n-heptylβ-D-thioglucoside; n-hexyl β-D-glucopyranoside;octanoyl-N-methylglucamide; n-octyl β-D-glucopyranoside;octanoyl-N-methylglucarmide; n-octyl-β-D-glucopyranoside; octylβ-D-thioglucopyranoside; and the like. Most of these surfactants areknown pharmaceutical excipients and are described in detail in theHandbook of Pharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and The Pharmaceutical Society of GreatBritain (The Pharmaceutical Press, 1986), specifically incorporated byreference for such disclosure.

The hydrophobic, water-insoluble and water-indispersible polymer orcopolymer may be chosen from biocompatible and biodegradable polymers,for example lactic or glycolic acid polymers and copolymers thereof, orpolylactic/polyethylene (or polypropylene) oxide copolymers, preferablywith molecular weights of between 1000 and 200,000, polyhydroxybutyricacid polymers, polylactones of fatty acids containing at least 12 carbonatoms, or polyanhydrides.

The nanoparticles may be obtained by coacervation, or by the techniqueof evaporation of solvent, from an aqueous dispersion or solution ofphospholipids and of an oleic acid salt into which is added animmiscible organic phase comprising the active principle and thehydrophobic, water-insoluble and water-indispersible polymer orcopolymer. The mixture is pre-emulsified and then subjected tohomogenization and evaporation of the organic solvent to obtain anaqueous suspension of very small-sized nanoparticles.

A variety of methods are optionally employed to fabricate the cytotoxicagent nanoparticles that are within the scope of the embodiments. Thesemethods include vaporization methods, such as free jet expansion, laservaporization, spark erosion, electro explosion and chemical vapordeposition; physical methods involving mechanical attrition (e.g.,“pearlmilling” technology, Elan Nanosystems), super critical CO2 andinterfacial deposition following solvent displacement. In oneembodiment, the solvent displacement method is used. The size ofnanoparticles produced by this method is sensitive to the concentrationof polymer in the organic solvent; the rate of mixing; and to thesurfactant employed in the process. Continuous flow mixers provide thenecessary turbulence to ensure small particle size. One type ofcontinuous flow mixing device that is optionally used to preparenanoparticles has been described (Hansen et al J Phys Chem 92, 2189-96,1988). In other embodiments, ultrasonic devices, flow throughhomogenizers or supercritical CO2 devices may be used to preparenanoparticles.

If suitable nanoparticle homogeneity is not obtained on directsynthesis, then size-exclusion chromatography is used to produce highlyuniform drug-containing particles that are freed of other componentsinvolved in their fabrication. Size-exclusion chromatography (SEC)techniques, such as gel-filtration chromatography, is used to separateparticle-bound cytotoxic agent or other pharmaceutical compound fromfree cytotoxic agent or other pharmaceutical compound, or to select asuitable size range of cytotoxic agent-containing nanoparticles. VariousSEC media, such as Superdex 200, Superose 6, Sephacryl 1000 arecommercially available and are employed for the size-based fractionationof such mixtures. Additionally, nanoparticles are optionally purified bycentrifugation, membrane filtration and by use of other molecularsieving devices, crosslinked gels/materials and membranes.

Auris-Acceptable Cyclodextrin and Other Stabilizing Formulations

In a specific embodiment, the auris-acceptable formulationsalternatively comprises a cyclodextrin. Cyclodextrins are cyclicoligosaccharides containing 6, 7, or 8 glucopyranose units, referred toas α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin respectively.Cyclodextrins have a hydrophilic exterior, which enhances water-soluble,and a hydrophobic interior which forms a cavity. In an aqueousenvironment, hydrophobic portions of other molecules often enter thehydrophobic cavity of cyclodextrin to form inclusion compounds.Additionally, cyclodextrins are also capable of other types ofnonbonding interactions with molecules that are not inside thehydrophobic cavity. Cyclodextrins have three free hydroxyl groups foreach glucopyranose unit, or 18 hydroxyl groups on α-cyclodextrin, 21hydroxyl groups on β-cyclodextrin, and 24 hydroxyl groups onγ-cyclodextrin. One or more of these hydroxyl groups can be reacted withany of a number of reagents to form a large variety of cyclodextrinderivatives, including hydroxypropyl ethers, sulfonates, andsulfoalkylethers. Shown below is the structure of β-cyclodextrin and thehydroxypropyl-β-cyclodextrin (HPβCD).

In some embodiments, the use of cyclodextrins in the pharmaceuticalcompositions described herein improves the solubility of the drug.Inclusion compounds are involved in many cases of enhanced solubility;however other interactions between cyclodextrins and insoluble compoundsalso improves solubility. Hydroxypropyl-β-cyclodextrin (HPβCD) iscommercially available as a pyrogen free product. It is a nonhygroscopicwhite powder that readily dissolves in water. HPβCD is thermally stableand does not degrade at neutral pH. Thus, cyclodextrins improve thesolubility of a therapeutic agent in a composition or formulation.Accordingly, in some embodiments, cyclodextrins are included to increasethe solubility of the auris-acceptable cytotoxic agents within theformulations described herein. In other embodiments, cyclodextrins inaddition serve as controlled release excipents within the formulationsdescribed herein.

By way of example only, cyclodextrin derivatives for use includeα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethylβ-cyclodextrin, hydroxypropyl γ-cyclodextrin, sulfated β-cyclodextrin,sulfated α-cyclodextrin, sulfobutyl ether β-cyclodextrin.

The concentration of the cyclodextrin used in the compositions andmethods disclosed herein varies according to the physiochemicalproperties, pharmacokinetic properties, side effect or adverse events,formulation considerations, or other factors associated with thetherapeutically active agent, or a salt or prodrug thereof, or with theproperties of other excipients in the composition. Thus, in certaincircumstances, the concentration or amount of cyclodextrin used inaccordance with the compositions and methods disclosed herein will vary,depending on the need. When used, the amount of cyclodextrins needed toincrease solubility of the cytotoxic agent and/or function as acontrolled release excipient in any of the formulations described hereinis selected using the principles, examples, and teachings describedherein.

Other stabilizers that are useful in the auris-acceptable formulationsdisclosed herein include, for example, fatty acids, fatty alcohols,alcohols, long chain fatty acid esters, long chain ethers, hydrophilicderivatives of fatty acids, polyvinyl pyrrolidones, polyvinyl ethers,polyvinyl alcohols, hydrocarbons, hydrophobic polymers,moisture-absorbing polymers, and combinations thereof. In someembodiments, amide analogues of stabilizers are also used. In furtherembodiments, the chosen stabilizer changes the hydrophobicity of theformulation (e.g., oleic acid, waxes), or improves the mixing of variouscomponents in the formulation (e.g., ethanol), controls the moisturelevel in the formula (e.g., PVP or polyvinyl pyrrolidone), controls themobility of the phase (substances with melting points higher than roomtemperature such as long chain fatty acids, alcohols, esters, ethers,amides etc. or mixtures thereof; waxes), and/or improves thecompatibility of the formula with encapsulating materials (e.g., oleicacid or wax). In another embodiment some of these stabilizers are usedas solvents/co-solvents (e.g., ethanol). In other embodiments,stabilizers are present in sufficient amounts to inhibit the degradationof the cytotoxic agent. Examples of such stabilizing agents, include,but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b)about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

Additional useful cytotoxic agent auris-acceptable formulations includeone or more anti-aggregation additives to enhance stability of cytotoxicagent formulations by reducing the rate of protein aggregation. Theanti-aggregation additive selected depends upon the nature of theconditions to which the cytotoxic agents, for example cytotoxic agentantibodies are exposed. For example, certain formulations undergoingagitation and thermal stress require a different anti-aggregationadditive than a formulation undergoing lyophilization andreconstitution. Useful anti-aggregation additives include, by way ofexample only, urea, guanidinium chloride, simple amino acids such asglycine or arginine, sugars, polyalcohols, polysorbates, polymers suchas polyethylene glycol and dextrans, alkyl saccharides, such as alkylglycoside, and surfactants.

Other useful formulations optionally include one or moreauris-acceptable antioxidants to enhance chemical stability whererequired. Suitable antioxidants include, by way of example only,ascorbic acid, methionine, sodium thiosulfate and sodium metabisulfite.In one embodiment, antioxidants are selected from metal chelatingagents, thiol containing compounds and other general stabilizing agents.

Still other useful compositions include one or more auris-acceptablesurfactants to enhance physical stability or for other purposes.Suitable nonionic surfactants include, but are not limited to,polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylenealkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

In some embodiments, the auris-acceptable pharmaceutical formulationsdescribed herein are stable with respect to compound degradation over aperiod of any of at least about 1 day, at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 1 week, at least about 2 weeks, at leastabout 3 weeks, at least about 4 weeks, at least about 5 weeks, at leastabout 6 weeks, at least about 7 weeks, at least about 8 weeks, at leastabout 3 months, at least about 4 months, at least about 5 months, or atleast about 6 months. In other embodiments, the formulations describedherein are stable with respect to compound degradation over a period ofat least about 1 week. Also described herein are formulations that arestable with respect to compound degradation over a period of at leastabout 1 month.

In other embodiments, an additional surfactant (co-surfactant) and/orbuffering agent is combined with one or more of the pharmaceuticallyacceptable vehicles previously described herein so that the surfactantand/or buffering agent maintains the product at an optimal pH forstability. Suitable co-surfactants include, but are not limited to: a)natural and synthetic lipophilic agents, e.g., phospholipids,cholesterol, and cholesterol fatty acid esters and derivatives thereof;b) nonionic surfactants, which include for example, polyoxyethylenefatty alcohol esters, sorbitan fatty acid esters (Spans),polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene (20)sorbitan monooleate (Tween 80), polyoxyethylene (20) sorbitanmonostearate (Tween 60), polyoxyethylene (20) sorbitan monolaurate(Tween 20) and other Tweens, sorbitan esters, glycerol esters, e.g.,Myrj and glycerol triacetate (triacetin), polyethylene glycols, cetylalcohol, cetostearyl alcohol, stearyl alcohol, polysorbate 80,poloxamers, poloxamines, polyoxyethylene castor oil derivatives (e.g.,Cremophor® RH40, Cremphor A25, Cremphor A20, Cremophor® EL) and otherCremophors, sulfosuccinates, alkyl sulphates (SLS); PEG glyceryl fattyacid esters such as PEG-8 glyceryl caprylate/caprate (Labrasol), PEG-4glyceryl caprylate/caprate (Labrafac Hydro WL 1219), PEG-32 glyceryllaurate (Gelucire 444/14), PEG-6 glyceryl mono oleate (Labrafil M 1944CS), PEG-6 glyceryl linoleate (Labrafil M 2125 CS); propylene glycolmono- and di-fatty acid esters, such as propylene glycol laurate,propylene glycol caprylate/caprate; Brij® 700, ascorbyl-6-palmitate,stearylamine, sodium lauryl sulfate, polyoxethyleneglyceroltriiricinoleate, and any combinations or mixtures thereof; c) anionicsurfactants include, but are not limited to, calciumcarboxymethylcellulose, sodium carboxymethylcellulose, sodiumsulfosuccinate, dioctyl, sodium alginate, alkyl polyoxyethylenesulfates, sodium lauryl sulfate, triethanolamine stearate, potassiumlaurate, bile salts, and any combinations or mixtures thereof; and d)cationic surfactants such as cetyltrimethylammonium bromide, andlauryldimethylbenzyl-ammonium chloride.

In a further embodiment, when one or more co-surfactants are utilized inthe auris-acceptable formulations of the present disclosure, they arecombined, e.g., with a pharmaceutically acceptable vehicle and ispresent in the final formulation, e.g., in an amount ranging from about0.1% to about 20%, from about 0.5% to about 10%.

In one embodiment, the surfactant has an HLB value of 0 to 20. Inadditional embodiments, the surfactant has an HLB value of 0 to 3, of 4to 6, of 7 to 9, of 8 to 18, of 13 to 15, of 10 to 18.

In one embodiment, diluents are also used to stabilize the cytotoxicagent or other pharmaceutical compounds because they provide a morestable environment. Salts dissolved in buffered solutions (which alsocan provide pH control or maintenance) are utilized as diluents,including, but not limited to a phosphate buffered saline solution. Inother embodiments, the gel formulation is isotonic with the endolymph orthe perilymph: depending on the portion of the cochlea that thecytotoxic agent formulation is targeted. Isotonic formulations areprovided by the addition of a tonicity agent. Suitable tonicity agentsinclude, but are not limited to any pharmaceutically acceptable sugar,salt or any combinations or mixtures thereof, such as, but not limitedto dextrose and sodium chloride. In further embodiments, the tonicityagents are present in an amount from about 100 mOsm/kg to about 500mOsm/kg. In some embodiments, the tonicity agent is present in an amountfrom about 200 mOsm/kg to about 400 mOsm/kg, from about 280 mOsm/kg toabout 320 mOsm/kg. The amount of tonicity agents will depend on thetarget structure of the pharmaceutical formulation, as described herein.

Useful tonicity compositions also include one or more salts in an amountrequired to bring osmolality of the composition into an acceptable rangefor the perilymph or the endolymph. Such salts include those havingsodium, potassium or ammonium cations and chloride, citrate, ascorbate,borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfiteanions; suitable salts include sodium chloride, potassium chloride,sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some embodiments, the auris-acceptable gel formulations disclosedherein alternatively or additionally contains preservatives to preventmicrobial growth. Suitable auris-acceptable preservatives for use in theenhanced viscosity formulations described herein include, but are notlimited to benzoic acid, boric acid, p-hydroxybenzoates, alcohols,quarternary compounds, stabilized chlorine dioxide, mercurials, such asmerfen and thiomersal, mixtures of the foregoing and the like.

In a further embodiment, the preservative is, by way of example only, anantimicrobial agent, within the auris-acceptable formulations presentedherein. In one embodiment, the formulation includes a preservative suchas by way of example only, methyl paraben, sodium bisulfite, sodiumthiosulfate, ascorbate, chorobutanol, thimerosal, parabens, benzylalcohol, phenylethanol and others. In another embodiment, the methylparaben is at a concentration of about 0.05% to about 1.0%, about 0.1%to about 0.2%. In a further embodiment, the gel is prepared by mixingwater, methylparaben, hydroxyethylcellulose and sodium citrate. In afurther embodiment, the gel is prepared by mixing water, methylparaben,hydroxyethylcellulose and sodium acetate. In a further embodiment, themixture is sterilized by autoclaving at 120° C. for about 20 minutes,and tested for pH, methylparaben concentration and viscosity beforemixing with the appropriate amount of the cytotoxic agent disclosedherein.

Suitable auris-acceptable water soluble preservatives which are employedin the drug delivery vehicle include sodium bisulfite, sodiumthiosulfate, ascorbate, chorobutanol, thimerosal, parabens, benzylalcohol, Butylated hydroxytoluene (BHT), phenylethanol and others. Theseagents are present, generally, in amounts of about 0.001% to about 5% byweight and, preferably, in the amount of about 0.01 to about 2% byweight. In some embodiments, auris-compatible formulations describedherein are free of preservatives.

Round Window Membrane Penetration Enhancers

In another embodiment, the formulation further comprises one or moreround window membrane penetration enhancers. Penetration across theround window membrane is enhanced by the presence of round windowmembrane penetration enhancers. Round window membrane penetrationenhancers are chemical entities that facilitate transport ofcoadministered substances across the round window membrane. Round windowmembrane penetration enhancers are grouped according to chemicalstructure. Surfactants, both ionic and non-ionic, such as sodium laurylsulfate, sodium laurate, polyoxyethylene-20-cetyl ether, laureth-9,sodium dodecylsulfate, dioctyl sodium sulfosuccinate,polyoxyethylene-9-lauryl ether (PLE), Tween® 80,nonylphenoxypolyethylene (NP-POE), polysorbates and the like, functionas round window membrane penetration enhancers. Bile salts (such assodium glycocholate, sodium deoxycholate, sodium taurocholate, sodiumtaurodihydrofusidate, sodium glycodihydrofusidate and the like), fattyacids and derivatives (such as oleic acid, caprylic acid, mono- anddi-glycerides, lauric acids, acylcholines, caprylic acids,acylcarnitines, sodium caprates and the like), chelating agents (such asEDTA, citric acid, salicylates and the like), sulfoxides (such asdimethyl sulfoxide (DMSO), decylmethyl sulfoxide and the like), andalcohols (such as ethanol, isopropanol, glycerol, propanediol and thelike) also function as round window membrane penetration enhancers.

In some embodiments, the auris acceptable penetration enhancer is asurfactant comprising an alkyl-glycoside wherein the alkyl glycoside istetradecyl-β-D-maltoside. In some embodiments, the auris acceptablepenetration enhancer is a surfactant comprising an alkyl-glycosidewherein the alkyl glycoside is dodecyl-maltoside. In certain instances,the penetration enhancing agent is a hyaluronidase. In certaininstances, a hyaluronidase is a human or bovine hyaluronidase. In someinstances, a hyaluronidase is a human hyaluronidase (e.g., hyaluronidasefound in human sperm, PH20 (Halozyme), Hyelenex® (Baxter International,Inc.)). In some instances, a hyaluronidase is a bovine hyaluronidase(e.g., bovine testicular hyaluronidase, Amphadase® (AmphastarPharmaceuticals), Hydase® (PrimaPharm, Inc). In some instances, ahyluronidase is an ovine hyaluronidase, Vitrase® (ISTA Pharmaceuticals).In certain instances, a hyaluronidase described herein is a recombinanthyaluronidase. In some instances, a hyaluronidase described herein is ahumanized recombinant hyaluronidase. In some instances, a hyaluronidasedescribed herein is a pegylated hyaluronidase (e.g., PEGPH20(Halozyme)). In addition, the peptide-like penetration enhancersdescribed in U.S. Pat. Nos. 7,151,191, 6,221,367 and 5,714,167, hereinincorporated by references for such disclosure, are contemplated as anadditional embodiment. These penetration enhancers are amino-acid andpeptide derviatives and enable drug absorption by passive transcellulardiffusion without affecting the integrity of membranes or intercellulartight junctions.

Round Window Membrane Permeable Liposomes

Liposomes or lipid particles may also be employed to encapsulate thecytotoxic agent formulations or compositions. Phospholipids that aregently dispersed in an aqueous medium form multilayer vesicles withareas of entrapped aqueous media separating the lipid layers.Sonication, or turbulent agitation, of these multilayer veiscles resultsin the formation of single layer vesicles, commonly referred to asliposomes, with sizes of about 10-1000 nm. These liposomes have manyadvantages as cytotoxic agents or other pharmaceutical agent carriers.They are biologically inert, biodegradable, non-toxic and non-antigenic.Liposomes are formed in various sizes and with varying compositions andsurface properties. Additionally, they are able to entrap a wide varietyof agents and release the agent at the site of liposome collapse.

Suitable phospholipids for use in auris-acceptable liposomes here are,for example, phosphatidyl cholines, ethanolamines and serines,sphingomyelins, cardiolipins, plasmalogens, phosphatidic acids andcerebrosides, in particular those which are soluble together with thecytotoxic agents herein in non-toxic, pharmaceutically acceptableorganic solvents. Preferred phospholipids are, for example, phosphatidylcholine, phosphatidyl ethanolmine, phosphatidyl serine, phosphatidylinositol, lysophosphatidyl choline, phosphatidyl glycerol and the like,and mixtures thereof especially lecithin, e.g. soya lecithin. The amountof phospholipid used in the present formulation range from about 10 toabout 30%, preferably from about 15 to about 25% and in particular isabout 20%.

Lipophilic additives may be employed advantageously to modifyselectively the characteristics of the liposomes. Examples of suchadditives include by way of example only, stearylamine, phosphatidicacid, tocopherol, cholesterol, cholesterol hemisuccinate and lanolinextracts. The amount of lipophilic additive used range from 0.5 to 8%,preferably from 1.5 to 4% and in particular is about 2%. Generally, theratio of the amount of lipophilic additive to the amount of phospholipidranges from about 1:8 to about 1:12 and in particular is about 1:10.Said phospholipid, lipophilic additive and the cytotoxic agent and otherpharmaceutical compounds are employed in conjunction with a non-toxic,pharmaceutically acceptable organic solvent system which dissolve saidingredients. Said solvent system not only must dissolve the cytotoxicagent completely, but it also has to allow the formulation of stablesingle bilayered liposomes. The solvent system comprisesdimethylisosorbide and tetraglycol (glycofurol, tetrahydrofurfurylalcohol polyethylene glycol ether) in an amount of about 8 to about 30%.In said solvent system, the ratio of the amount of dimethylisosorbide tothe amount of tetraglycol range from about 2:1 to about 1:3, inparticular from about 1:1 to about 1:2.5 and preferably is about 1:2.The amount of tetraglycol in the final composition thus vary from 5 to20%, in particular from 5 to 15% and preferably is approximately 10%.The amount of dimethylisosorbide in the final composition thus rangefrom 3 to 10%, in particular from 3 to 7% and preferably isapproximately 5%.

The term “organic component” as used hereinafter refers to mixturescomprising said phospholipid, lipophilic additives and organic solvents.The cytotoxic agent may be dissolved in the organic component, or othermeans to maintain full activity of the agent. The amount of cytotoxicagent in the final formulation may range from 0.1 to 5.0%. In addition,other ingredients such as anti-oxidants may be added to the organiccomponent. Examples include tocopherol, butylated hydroxyanisole,butylated hydroxytoluene, ascorbyl palmitate, ascorbyl oleate and thelike.

Liposomal formulations are alternatively prepared, for cytotoxic agentsor other pharmaceutical agents that are moderately heat-resistant, by(a) heating the phospholipid and the organic solvent system to about60-80° C. in a vessel, dissolving the active ingredient, then adding anyadditional formulating agents, and stirring the mixture until completedissolution is obtained; (b) heating the aqueous solution to 90-95° C.in a second vessel and dissolving the preservatives therein, allowingthe mixture to cool and then adding the remainder of the auxiliaryformulating agents and the remainder of the water, and stirring themixture until complete dissolution is obtained; thus preparing theaqueous component; (c) transferring the organic phase directly into theaqueous component, while homogenizing the combination with a highperformance mixing apparatus, for example, a high-shear mixer; and (d)adding a viscosity enhancing agent to the resulting mixture whilefurther homogenizing. The aqueous component is optionally placed in asuitable vessel which is equipped with a homogenizer and homogenizationis effected by creating turbulence during the injection of the organiccomponent. Any mixing means or homogenizer which exerts high shearforces on the mixture may be employed. Generally, a mixer capable ofspeeds from about 1,500 to 20,000 rpm, in particular from about 3,000 toabout 6,000 rpm may be employed. Suitable viscosity enhancing agents foruse in process step (d) are for example, xanthan gum, hydroxypropylcellulose, hydroxypropyl methylcellulose or mixtures thereof. The amountof viscosity enhancing agent depends on the nature and the concentrationof the other ingredients and in general ranges from about 0.5 to 2.0%,or approximately 1.5%. In order to prevent degradation of the materialsused during the preparation of the liposomal formulation, it isadvantageous to purge all solutions with an inert gas such as nitrogenor argon, and to conduct all steps under an inert atmosphere. Liposomesprepared by the above described method usually contain most of theactive ingredient bound in the lipid bilayer and separation of theliposomes from unencapsulated material is not required.

In other embodiments, the auris-acceptable formulations, including gelformulations and viscosity-enhanced formulations, further includeexcipients, other medicinal or pharmaceutical agents, carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts, solubilizers, an antifoaming agent,an antioxidant, a dispersing agent, a wetting agent, a surfactant, andcombinations thereof.

Suitable carriers for use in an auris-acceptable formulation describedherein include, but are not limited to, any pharmaceutically acceptablesolvent compatible with the targeted auris structure's physiologicalenvironment. In other embodiments, the base is a combination of apharmaceutically acceptable surfactant and solvent.

In some embodiments, other excipients include, sodium stearyl fumarate,diethanolamine cetyl sulfate, isostearate, polyethoxylated castor oil,nonoxyl 10, octoxynol 9, sodium lauryl sulfate, sorbitan esters(sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate,sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate,sorbitan tristearate, sorbitan laurate, sorbitan oleate, sorbitanpalmitate, sorbitan stearate, sorbitan dioleate, sorbitansesqui-isostearate, sorbitan sesquistearate, sorbitan tri-isostearate),lecithin pharmaceutical acceptable salts thereof and combinations ormixtures thereof.

In other embodiments, the carrier is a polysorbate. Polysorbates arenonionic surfactants of sorbitan esters. Polysorbates useful in thepresent disclosure include, but are not limited to polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80 (Tween 80) and anycombinations or mixtures thereof. In further embodiments, polysorbate 80is utilized as the pharmaceutically acceptable carrier.

In one embodiment, water-soluble glycerin-based auris-acceptableenhanced viscosity formulations utilized in the preparation ofpharmaceutical delivery vehicles comprise at least one cytotoxic agentcontaining at least about 0.1% of the water-soluble glycerin compound ormore. In some embodiments, the percentage of cytotoxic agent is variedbetween about 1% and about 95%, between about 5% and about 80%, betweenabout 10% and about 60% or more of the weight or volume of the totalpharmaceutical formulation. In some embodiments, the amount of thecompound(s) in each therapeutically useful cytotoxic agent formulationis prepared in such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations arecontemplated herein.

If desired, the auris-acceptable pharmaceutical gels also containco-solvents, preservatives, cosolvents, ionic strength and osmolalityadjustors and other excipients in addition to buffering agents. Suitableauris-acceptable water soluble buffering agents are alkali or alkalineearth metal carbonates, phosphates, bicarbonates, citrates, borates,acetates, succinates and the like, such as sodium phosphate, citrate,borate, acetate, bicarbonate, carbonate and tromethamine (TRIS). Theseagents are present in amounts sufficient to maintain the pH of thesystem at 7.4±0.2 and preferably, 7.4. As such, the buffering agent isas much as 5% on a weight basis of the total composition.

Cosolvents are used to enhance cytotoxic agent solubility, however, somecytotoxic agents or other pharmaceutical compounds are insoluble. Theseare often suspended in the polymer vehicle with the aid of suitablesuspending or viscosity enhancing agents.

Moreover, some pharmaceutical excipients, diluents or carriers arepotentially ototoxic. For example, benzalkonium chloride, a commonpreservative, is ototoxic and therefore potentially harmful ifintroduced into the vestibular or cochlear structures. In formulating acontrolled release cytotoxic agent formulation, it is advised to avoidor combine the appropriate excipients, diluents or carriers to lessen oreliminate potential ototoxic components from the formulation, or todecrease the amount of such excipients, diluents or carriers.Optionally, a controlled release cytotoxic agent formulation includesotoprotective agents, such as antioxidants, alpha lipoic acid, calicum,fosfomycin or iron chelators, to counteract potential ototoxic effectsthat may arise from the use of specific therapeutic agents orexcipients, diluents or carriers.

Thof therapeutically acceptable otic formulations:

Example Formulation Example Characteristics Chitosan tunable degradationof matrix in vitro glycerophosphate (CGP) tunable TACE inhibitor releasein vitro: e.g., ~50% of drug released after 24 hrs biodegradablecompatible with drug delivery to the inner ear suitable formacromolecules and hydrophobic drugs PEG-PLGA-PEG triblock tunable highstability: e.g., maintains mechanical integrity > polymers 1 month invitro tunable fast release of hydrophilic drugs: e.g., ~50% of drugreleased after 24 hrs, and remainder released over ~5 days tunable slowrelease of hydrophobic drugs: e.g., ~80% released after 8 weeksbiodegradable subcutaneous injection of solution: e.g., gel forms withinseconds and is intact after 1 month PEO-PPO-PEO triblock Tunable sol-geltransition temperature: e.g., decreases copolymers (e.g., withincreasing F127 concentration Pluronic or Poloxameres) (e.g., F127)Chitosan CGP formulation tolerates liposomes: e.g., up to 15 uM/mlglycerophosphate with liposomes. drug-loaded liposomes liposomes tunablyreduce drug release time (e.g., up to 2 weeks in vitro). increase inliposome diameter optionally reduces drug release kinetics (e.g.,liposome size between 100 and 300 nm) release parameters are controlledby changing composition of liposomes

The formulations disclosed herein alternatively encompass anotoprotectant agent in addition to the at least one active agent and/orexcipients, including but not limited to such agents as antioxidants,alpha lipoic acid, calcium, fosfomycin or iron chelators, to counteractpotential ototoxic effects that may arise from the use of specifictherapeutic agents or excipients, diluents or carriers.

Modes of Treatment

Dosing Methods and Schedules

Drugs delivered to the inner ear have been administered systemically viaoral, intravenous or intramuscular routes. However, systemicadministration for pathologies local to the inner ear increases thelikelihood of systemic toxicities and adverse side effects and creates anon-productive distribution of drug in which high levels of drug arefound in the serum and correspondingly lower levels are found at theinner ear.

Intratympanic injection of therapeutic agents is the technique ofinjecting a therapeutic agent behind the tympanic membrane into themiddle and/or inner ear. In one embodiment, the formulations describedherein are administered directly onto the round window membrane viatranstympanic injection. In another embodiment, the cytotoxic agentauris-acceptable formulations described herein are administered onto theround window membrane via a non-transtympanic approach to the inner ear.In additional embodiments, the formulation described herein isadministered onto the round window membrane via a surgical approach tothe round window membrane comprising modification of the cristafenestrae cochleae.

In one embodiment the delivery system is a syringe and needle apparatusthat is capable of piercing the tympanic membrane and directly accessingthe round window membrane or crista fenestrae cochleae of the aurisinterna. In some embodiments, the needle on the syringe is wider than a18 gauge needle. In another embodiment, the needle gauge is from 18gauge to 31 gauge. In a further embodiment, the needle gauge is from 25gauge to 30 gauge. Depending upon the thickness or viscosity of thecytotoxic agent compositions or formulations, the gauge level of thesyringe or hypodermic needle may be varied accordingly. In anotherembodiment, the internal diameter of the needle can be increased byreducing the wall thickness of the needle (commonly referred as thinwall or extra thin wall needles) to reduce the possibility of needleclogging while maintaining an adequate needle gauge.

In another embodiment, the needle is a hypodermic needle used forinstant delivery of the gel formulation. The hypodermic needle may be asingle use needle or a disposable needle. In some embodiments, a syringemay be used for delivery of the pharmaceutically acceptable gel-basedcytotoxic agent-containing compositions as disclosed herein wherein thesyringe has a press-fit (Luer) or twist-on (Luer-lock) fitting. In oneembodiment, the syringe is a hypodermic syringe. In another embodiment,the syringe is made of plastic or glass. In yet another embodiment, thehypodermic syringe is a single use syringe. In a further embodiment, theglass syringe is capable of being sterilized. In yet a furtherembodiment, the sterilization occurs through an autoclave. In anotherembodiment, the syringe comprises a cylindrical syringe body wherein thegel formulation is stored before use. In other embodiments, the syringecomprises a cylindrical syringe body wherein the cytotoxic agentpharmaceutically acceptable gel-based compositions as disclosed hereinis stored before use which conveniently allows for mixing with asuitable pharmaceutically acceptable buffer. In other embodiments, thesyringe may contain other excipients, stabilizers, suspending agents,diluents or a combination thereof to stabilize or otherwise stably storethe cytotoxic agent or other pharmaceutical compounds contained therein.

In some embodiments, the syringe comprises a cylindrical syringe bodywherein the body is compartmentalized in that each compartment is ableto store at least one component of the auris-acceptable cytotoxic agentgel formulation. In a further embodiment, the syringe having acompartmentalized body allows for mixing of the components prior toinjection into the auris media or auris interna. In other embodiments,the delivery system comprises multiple syringes, each syringe of themultiple syringes contains at least one component of the gel formulationsuch that each component is pre-mixed prior to injection or is mixedsubsequent to injection. In a further embodiment, the syringes disclosedherein comprise at least one reservoir wherein the at least onereservoir comprises a cytotoxic agent, or a pharmaceutically acceptablebuffer, or a viscosity enhancing agent, such as a gelling agent or acombination thereof. Commercially available injection devices areoptionally employed in their simplest form as ready-to-use plasticsyringes with a syringe barrel, needle assembly with a needle, plungerwith a plunger rod, and holding flange, to perform an intratympanicinjection.

In some embodiments, the delivery device is an apparatus designed foradministration of therapeutic agents to the middle and/or inner ear. Byway of example only: GYRUS Medical Gmbh offers micro-otoscopes forvisualization of and drug delivery to the round window niche; Arenberghas described a medical treatment device to deliver fluids to inner earstructures in U.S. Pat. Nos. 5,421,818; 5,474,529; and 5,476,446, eachof which is incorporated by reference herein for such disclosure. U.S.patent application Ser. No. 08/874,208, which is incorporated herein byreference for such disclosure, describes a surgical method forimplanting a fluid transfer conduit to deliver therapeutic agents to theinner ear. U.S. Patent Application Publication 2007/0167918, which isincorporated herein by reference for such disclosure, further describesa combined otic aspirator and medication dispenser for intratympanicfluid sampling and medicament application.

The formulations described herein, and modes of administration thereof,are also applicable to methods of direct instillation or perfusion ofthe inner ear compartments. Thus, the formulations described herein areuseful in surgical procedures including, by way of non-limitingexamples, cochleostomy, labyrinthotomy, mastoidectomy, stapedectomy,endolymphatic sacculotomy or the like.

The auris-acceptable compositions or formulations containing thecytotoxic agent compound(s) described herein are administered forprophylactic and/or therapeutic treatments. In therapeutic applications,the cytotoxic agent compositions are administered to a patient alreadysuffering from an autoimmune disease, condition or disorder, in anamount sufficient to cure or at least partially arrest the symptoms ofthe disease, disorder or condition. Amounts effective for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the cytotoxic agent compoundsmay be administered chronically, that is, for an extended period oftime, including throughout the duration of the patient's life in orderto ameliorate or otherwise control or limit the symptoms of thepatient's disease or condition.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the cytotoxic agent compounds may begiven continuously; alternatively, the dose of drug being administeredmay be temporarily reduced or temporarily suspended for a certain lengthof time (i.e., a “drug holiday”). The length of the drug holiday variesbetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days,and 365 days. The dose reduction during a drug holiday may be from10%-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

Once improvement of the patient's otic conditions has occurred, amaintenance cytotoxic agent dose is administered if necessary.Subsequently, the dosage or the frequency of administration, or both, isoptionally reduced, as a function of the symptoms, to a level at whichthe improved disease, disorder or condition is retained. In certainembodiments, patients require intermittent treatment on a long-termbasis upon any recurrence of symptoms.

The amount of cytotoxic agent that will correspond to such an amountwill vary depending upon factors such as the particular compound,disease condition and its severity, according to the particularcircumstances surrounding the case, including, e.g., the specificcytotoxic agent being administered, the route of administration, theautoimmune condition being treated, the target area being treated, andthe subject or host being treated. In general, however, doses employedfor adult human treatment will typically be in the range of 0.02-50 mgper administration, preferably 1-15 mg per administration. The desireddose is presented in a single dose or as divided doses administeredsimultaneously (or over a short period of time) or at appropriateintervals.

In some embodiments, the initial administration is a particularcytotoxic agent and the subsequent administration a differentformulation or cytotoxic agent.

Pharmacokinetics of Controlled Release Formulations

In one embodiment, the formulations disclosed herein additionallyprovides an immediate release of a cytotoxic agent from the composition,or within 1 minute, or within 5 minutes, or within 10 minutes, or within15 minutes, or within 30 minutes, or within 60 minutes or within 90minutes. In other embodiments, a therapeutically effective amount of atleast one cytotoxic agent is released from the composition immediately,or within 1 minute, or within 5 minutes, or within 10 minutes, or within15 minutes, or within 30 minutes, or within 60 minutes or within 90minutes. In certain embodiments the composition comprises anauris-pharmaceutically acceptable gel formulation providing immediaterelease of at least one cytotoxic agent. Additional embodiments of theformulation may also include an agent that enhances the viscosity of theformulations included herein.

In other or further embodiments, the formulation provides an extendedrelease formulation of at least one cytotoxic agent. In certainembodiments, diffusion of at least one cytotoxic agent from theformulation occurs for a time period exceeding 5 minutes, or 15 minutes,or 30 minutes, or 1 hour, or 4 hours, or 6 hours, or 12 hours, or 18hours, or 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days,or 7 days, or 10 days, or 12 days, or 14 days, or 18 days, or 21 days,or 25 days, or 30 days, or 45 days, or 2 months or 3 months or 4 monthsor 5 months or 6 months or 9 months or 1 year. In other embodiments, atherapeutically effective amount of at least one cytotoxic agent isreleased from the formulation for a time period exceeding 5 minutes, or15 minutes, or 30 minutes, or 1 hour, or 4 hours, or 6 hours, or 12hours, or 18 hours, or 1 day, or 2 days, or 3 days, or 4 days, or 5days, or 6 days, or 7 days, or 10 days, or 12 days, or 14 days, or 18days, or 21 days, or 25 days, or 30 days, or 45 days, or 2 months or 3months or 4 months or 5 months or 6 months or 9 months or 1 year.

In other embodiments, the formulation provides both an immediate releaseand an extended release formulation of a cytotoxic agent. In yet otherembodiments, the formulation contains a 0.25:1 ratio, or a 0.5:1 ratio,or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5ratio, or a 1:7 ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratioof immediate release and extended release formulations. In a furtherembodiment the formulation provides an immediate release of a firstcytotoxic agent and an extended release of a second cytotoxic agent orother therapeutic agent. In yet other embodiments, the formulationprovides an immediate release and extended release formulation of atleast one cytotoxic agent, and at least one therapeutic agent. In someembodiments, the formulation provides a 0.25:1 ratio, or a 0.5:1 ratio,or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5ratio, or a 1:7 ratio, or a 1:10 ratio, or a 1:15 ratio, or a 1:20 ratioof immediate release and extended release formulations of a firstcytotoxic agent and second therapeutic agent, respectively.

In a specific embodiment the formulation provides a therapeuticallyeffective amount of at least one cytotoxic agent at the site of diseasewith essentially no systemic exposure. In an additional embodiment theformulation provides a therapeutically effective amount of at least onecytotoxic agent at the site of disease with essentially no detectablesystemic exposure. In other embodiments, the formulation provides atherapeutically effective amount of at least one cytotoxic agent at thesite of disease with little or no detectable systemic exposure.

The combination of immediate release, delayed release and/or extendedrelease cytotoxic agent compositions or formulations may be combinedwith other pharmaceutical agents, as well as the excipients, diluents,stabilizers, tonicity agents and other components disclosed herein. Assuch, depending upon the cytotoxic agent used, the thickness orviscosity desired, or the mode of delivery chosen, alternative aspectsof the embodiments disclosed herein are combined with the immediaterelease, delayed release and/or extended release embodimentsaccordingly.

In certain embodiments, the pharmacokinetics of the cytotoxic agentformulations described herein are determined by injecting theformulation on or near the round window membrane of a test animal(including by way of example, a guinea pig or a chinchilla). At adetermined period of time (e.g., 6 hours, 12 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, and 7 days for testing thepharmacokinetics of a formulation over a 1 week period), the test animalis euthanized and a 5 mL sample of the perilymph fluid is tested. Theinner ear removed and tested for the presence of the cytotoxic agent. Asneeded, the level of cytotoxic agent is measured in other organs. Inaddition, the systemic level of the cytotoxic agent is measured bywithdrawing a blood sample from the test animal. In order to determinewhether the formulation impedes hearing, the hearing of the test animalis optionally tested.

Alternatively, an inner ear is provided (as removed from a test animal)and the migration of the cytotoxic agent is measured. As yet anotheralternative, an in vitro model of a round window membrane is providedand the migration of the cytotoxic agent is measured.

Kits/Articles of Manufacture

The disclosure also provides kits for preventing, treating orameliorating the symptoms of a disease or disorder in a mammal. Suchkits generally will comprise one or more of the cytotoxic agentcontrolled-release compositions or devices disclosed herein, andinstructions for using the kit. The disclosure also contemplates the useof one or more of the cytotoxic agent controlled-release compositions,in the manufacture of medicaments for treating, abating, reducing, orameliorating the symptoms of a disease, dysfunction, or disorder in amammal, such as a human that has, is suspected of having, or at risk fordeveloping an inner ear disorder.

In some embodiments, kits include a carrier, package, or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) including one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In other embodiments, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arealso presented herein. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558and 5,033,252. Examples of pharmaceutical packaging materials include,but are not limited to, blister packs, bottles, tubes, inhalers, pumps,bags, vials, containers, syringes, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. A wide array of cytotoxic agent formulations compositionsprovided herein are contemplated as are a variety of treatments for anydisease, disorder, or condition that would benefit by controlled releaseadministration of a cytotoxic agent to the inner ear.

In some embodiments, a kit includes one or more additional containers,each with one or more of various materials (such as reagents, optionallyin concentrated form, and/or devices) desirable from a commercial anduser standpoint for use of a formulation described herein. Non-limitingexamples of such materials include, but not limited to, buffers,diluents, filters, needles, syringes; carrier, package, container, vialand/or tube labels listing contents and/or instructions for use andpackage inserts with instructions for use. A set of instructions isoptionally included. In a further embodiment, a label is on orassociated with the container. In yet a further embodiment, a label ison a container when letters, numbers or other characters forming thelabel are attached, molded or etched into the container itself; a labelis associated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Inother embodiments a label is used to indicate that the contents are tobe used for a specific therapeutic application. In yet anotherembodiment, a label also indicates directions for use of the contents,such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. In another embodiment, the packfor example contains metal or plastic foil, such as a blister pack. In afurther embodiment, the pack or dispenser device is accompanied byinstructions for administration. In yet a further embodiment, the packor dispenser is also accompanied with a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the drug for human orveterinary administration. In another embodiment, such notice, forexample, is the labeling approved by the U.S. Food and DrugAdministration for prescription drugs, or the approved product insert.In yet another embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES Example 1 Preparation of a Methotrexate Thermoreversible GelFormulation

Quantity (mg/g of Ingredient formulation) Methotrexate 1.0 Methylparaben1.0 HPMC 10.0 Poloxamer 407 180.0 TRIS HCl buffer (0.1 M) 808.0

A 10-g batch of gel formulation containing 0.1% of the cytotoxic agentmethotrexate is prepared by suspending 1.80 g of Poloxamer 407 (BASFCorp.) in 5.00 g of TRIS HCl buffer (0.1 M) and the components are mixedunder agitation overnight at 4° C. to ensure complete dissolution. Thehydroxypropylmethylcellulose (100.0 mg), methylparaben (10 mg) andadditional TRIS HCl buffer (0.1 M) (3.08 g) are added and furtherstirring allowed until complete dissolution is observed. Methotrexate(10 mg) is added and mixed in order to solubilize. The mixture ismaintained below room temperature until use.

Example 2 Preparation of a Methotrexate Mucoadhesive, ThermoreversibleGel Formulation

Quantity (mg/g of Ingredient formulation) Methotrexate 10.0Methylparaben 1.0 HPMC 10.0 Carbopol 934P 2.0 Poloxamer 407 180.0 TRISHCl buffer (0.1 M) 797.0

A 10-g batch of a mucoadhesive, gel formulation containing 1.0% of thecytotoxic agent methotrexate is prepared by suspending 20.0 mg ofCarbopol 934P and 1.80 g of Poloxamer 407 (BASF Corp.) in 5.00 g of TRISHCl buffer (0.1 M) and the components are mixed under agitationovernight at 4° C. to ensure complete dissolution. Thehydroxypropylmethylcellulose (100.0 mg), methylparaben (10 mg) andadditional TRIS HCl buffer (0.1 M) (2.97 g) are added and furtherstirring allowed until complete dissolution is observed. Themethotrexate (100 mg) is added and mixed in order to solubilize. Themixture is maintained below room temperature until use.

Example 3 Preparation of a Cyclophosphamide Mucoadhesive-BasedFormulation

Quantity (mg/g of Ingredient formulation) Cyclophosphamide 10.0 Paraffinoil 200 Trihydroxystearate 10 Cetyl dimethicon copolyol 30 Water qs ad1000 Phosphate buffer pH 7.4 qs pH 7.4

The cream-type formulation is first prepared by gently mixingcyclophosphamide with an organic solvent. A second system is prepared bymixing paraffin oil, trihydroxystearate and cetyl dimethicon copolyolwith warming to 60° C. Upon cooling to room temperature, the lipidsystem is mixed with the aqueous phase for 30 minutes.

Example 4 Preparation of a Cyclophosphamide Mucoadhesive,Thermoreversible Gel Formulation

Quantity (mg/g of Ingredient formulation) Methotrexate 1.0 Methylparaben1.0 Poloxamer 407 180.0 Carbopol 934P 2.0 TRIS HCl buffer (0.1 M) 316.0

The Carbopol 934P and Poloxamer 407 (BASF Corp.) is first suspended inthe TRIS HCl buffer (0.1 M) and the components are mixed under agitationovernight at 4° C. to ensure complete dissolution. The methylparaben isadded and further stirring allowed until complete dissolution isobserved. The methotrexate is mixed in while maintaining stirring toproduce a 0.2% methotrexate mucoadhesive, thermoreversible gelformulation. The mixture is maintained below room temperature until use.

Viscosity determinations of the pharmaceutical compositions describedherein are performed at room temperature and 37° C. and are made using aBrookfield (spindle and cup) viscometer at 20 rpm.

Example 5 Preparation of a Thalidomide Gel Formulation

Quantity (mg/g of Ingredient formulation) thalidomide 1.0 chitosan 20.0Glycerophosphate disodium 80.0 water 899

A 5 ml solution of acetic acid is titrated to a pH of about 4.0. Thechitosan is added to achieve a pH of about 5.5. The thalidomide is thendissolved in the chitosan solution. This solution is sterilized byfiltration. A 5 ml aqueous solution of glycerophosphate disodium is alsoprepared and sterilized. The two solutions are mixed and within 2 h at37° C., the desired gel is formed.

Example 6 Effect of pH on Degradation Products for Autoclaved 17%Poloxamer 407NF/2% Otic Agent in PBS Buffer

A stock solution of a 17% poloxamer 407/2% otic agent is prepared bydissolving 351.4 mg of sodium chloride (Fisher Scientific), 302.1 mg ofsodium phosphate dibasic anhydrous (Fisher Scientific), 122.1 mg ofsodium phosphate monobasic anhydrous (Fisher Scientific) and anappropriate amount of an otic agent with 79.3 g of sterile filtered DIwater. The solution is cooled down in a ice chilled water bath and then17.05 g of poloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into thecold solution while mixing. The mixture is further mixed until thepoloxamer is completely dissolved. The pH for this solution is measured.

17% poloxamer 407/2% otic agent in PBS pH of 5.3. Take an aliquot(approximately 30 mL) of the above solution and adjust the pH to 5.3 bythe addition of 1 M HCl.

17% poloxamer 407/2% otic agent in PBS pH of 8.0. Take an aliquot(approximately 30 mL) of the above stock solution and adjust the pH to8.0 by the addition of 1 M NaOH.

A PBS buffer (pH 7.3) is prepared by dissolving 805.5 mg of sodiumchloride (Fisher Scientific), 606 mg of sodium phosphate dibasicanhydrous (Fisher Scientific), 247 mg of sodium phosphate monobasicanhydrous (Fisher Scientific), then QS to 200 g with sterile filtered DIwater.

A 2% solution of an otic agent in PBS pH 7.3 is prepared by dissolvingan appropriate amount of the otic agent in the PBS buffer and QS to 10 gwith PBS buffer.

One mL samples are individually placed in 3 mL screw cap glass vials(with rubber lining) and closed tightly. The vials are placed in aMarket Forge-sterilmatic autoclave (settings, slow liquids) andsterilized at 250° F. for 15 minutes. After the autoclave the samplesare left to cool down to room temperature and then placed inrefrigerator. The samples are homogenized by mixing the vials whilecold.

Appearance (e.g., discoloration and/or precipitation) is observed andrecorded. HPLC analysis is performed using an Agilent 1200 equipped witha Luna C18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrilegradient (1-10 min) of (water-acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30 Lof sample and dissolved with 1.5 mL of a 1:1 acetonitrile water mixture.Purity of the otic agent in the autoclaved samples is recorded.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to the procedure above, are tested usingthe above procedure to determine the effect of pH on degradation duringthe autoclaving step.

Example 7 Effect of Autoclaving on the Release Profile and Viscosity ofa 17% Poloxamer 407NF/2% Otic Agent in PBS

An aliquot of a sample (autoclaved and not autoclaved) is evaluated forrelease profile and viscosity measurement to evaluate the impact of heatsterilization on the properties of the gel.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm). 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL is placed intoreservoir and shaken using a Labline orbit shaker at 70 rpm. Samples aretaken every hour (0.1 mL withdrawn and replace with warm buffer).Samples are analyzed for poloxamer concentration by UV at 624 nm usingthe cobalt thiocyanate method, against an external calibration standardcurve. In brief, 20 μL of the sample is mixed with 1980 μL of a 15 mMcobalt thiocyanate solution and absorbance measured at 625 nm, using aEvolution 160 UV/Vis spectrophotometer (Thermo Scientific).

The released otic agent is fitted to the Korsmeyer-Peppas equation

$\frac{Q}{Q_{x}} = {{kt}^{n} + b}$

where Q is the amount of otic agent released at time t, Q_(α) is theoverall released amount of otic agent, k is a release constant of thenth order, n is a dimensionless number related to the dissolutionmechanism and b is the axis intercept, characterizing the initial burstrelease mechanism wherein n=1 characterizes an erosion controlledmechanism. The mean dissolution time (MDT) is the sum of differentperiods of time the drug molecules stay in the matrix before release,divided by the total number of molecules and is calculated by:

${MDT} = \frac{{nk}^{- 1}n}{n + 1}$

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to the procedures described above, aretested using the procedure described above to determine Tgel.

Example 8 Effect of Addition of a Secondary Polymer on the DegradationProducts and Viscosity of a Formulation Containing 2% Otic Agent and 17%Poloxamer 407NF after Heat Sterilization (Autoclaving)

Solution A. A solution of pH 7.0 comprising sodiumcarboxymethylcellulose (CMC) in PBS buffer is prepared by dissolving178.35 mg of sodium chloride (Fisher Scientific), 300.5 mg of sodiumphosphate dibasic anhydrous (Fisher Scientific), 126.6 mg of sodiumphosphate monobasic anhydrous (Fisher Scientific) dissolved with 78.4 ofsterile filtered DI water, then 1 g of Blanose 7M65 CMC (Hercules,viscosity of 5450 cP @ 2%) is sprinkled into the buffer solution andheated to aid dissolution, and the solution is then cooled down.

A solution of pH 7.0 comprising 17% poloxamer 407NF/1% CMC/2% otic agentin PBS buffer is made by cooling down 8.1 g of solution A in a icechilled water bath and then adding an appropriate amount of an oticagent followed by mixing 1.74 g of poloxamer 407NF (Spectrum Chemicals)is sprinkled into the cold solution while mixing. The mixture is furthermixed until all the poloxamer is completely dissolved.

Two mL of the above sample is placed in a 3 mL screw cap glass vial(with rubber lining) and closed tightly. The vial is placed in a MarketForge-sterilmatic autoclave (settings, slow liquids) and sterilized at250° F. for 25 minutes. After autoclaving the sample is left to cooldown to room temperature and then placed in refrigerator. The sample ishomogenized by mixing while the vials are cold.

Precipitation or discoloration are observed after autoclaving. HPLCanalysis is performed using an Agilent 1200 equipped with a Luna C18(2)3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrile gradient(1-10 min) of (water-acetonitrile mixture containing 0.05% TFA), for atotal run of 15 minutes. Samples are diluted by taking 30 μL of sampleand dissolving with 1.5 mL of a 1:1 acetonitrile water mixture. Purityof the otic agent in the autoclaved samples is recorded.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Dissolution is performed at 37° C. for the non-autoclaved sample insnapwells (6.5 mm diameter polycarbonate membrane with a pore size of0.4 μm), 0.2 mL of gel is placed into snapwell and left to harden, then0.5 mL is placed into reservoir and shaken using a Labline orbit shakerat 70 rpm. Samples are taken every hour (0.1 mL withdrawn and replacedwith warm buffer). Samples are analyzed for otic agent concentration byUV at 245 nm, against an external calibration standard curve.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, are tested using the above procedure to determine theeffect addition of a secondary polymer on the degradation products andviscosity of a formulation containing 2% otic agent and 17% poloxamer407NF after heat sterilization (autoclaving).

Example 9 Effect of Buffer Type on the Degradation Products forFormulations Containing Poloxamer 407NF after Heat Sterilization(Autoclaving)

A TRIS buffer is made by dissolving 377.8 mg of sodium chloride (FisherScientific), and 602.9 mg of Tromethamine (Sigma Chemical Co.) then QSto 100 g with sterile filtered DI water, pH is adjusted to 7.4 with 1MHCl.

Stock Solution Containing 25% Poloxamer 407 Solution in TRIS Buffer:

Weigh 45 g of TRIS buffer, chill in an ice chilled bath then sprinkleinto the buffer, while mixing, 15 g of poloxamer 407 NF (SpectrumChemicals). The mixture is further mixed until all the poloxamer iscompletely dissolved.

A series of formulations is prepared with the above stock solution. Anappropriate amount of otic agent (or salt or prodrug thereof) and/orotic agent as micronized/coated/liposomal particles (or salt or prodrugthereof) is used for all experiments.

Stock Solution (pH 7.3) Containing 25% Poloxamer 407 Solution in PBSBuffer:

PBS buffer described above is used. Dissolve 704 mg of sodium chloride(Fisher Scientific), 601.2 mg of sodium phosphate dibasic anhydrous(Fisher Scientific), 242.7 mg of sodium phosphate monobasic anhydrous(Fisher Scientific) with 140.4 g of sterile filtered DI water. Thesolution is cooled down in an ice chilled water bath and then 50 g ofpoloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into the cold solutionwhile mixing. The mixture is further mixed until the poloxamer iscompletely dissolved.

A series of formulations is prepared with the above stock solution. Anappropriate amount of otic agent (or salt or prodrug thereof) and/orotic agent as micronized/coated/liposomal particles (or salt or prodrugthereof) is used for all experiments.

Tables 2 and 3 list samples prepared using the procedures describedabove. An appropriate amount of otic agent is added to each sample toprovide a final concentration of 2% otic agent in the sample.

TABLE 2 Preparation of samples containing TRIS buffer 25% Stock SolutionTRIS Buffer Sample pH (g) (g) 20% P407/2% otic agent/TRIS 7.45 8.01 1.8218% P407/2% otic agent/TRIS 7.45 7.22 2.61 16% P407/2% otic agent/TRIS7.45 6.47 3.42 18% P407/2% otic agent/TRIS 7.4 7.18 2.64 4% oticagent/TRIS 7.5 — 9.7 2% otic agent/TRIS 7.43 — 5 1% otic agent/TRIS 7.35— 5 2% otic agent/TRIS 7.4 — 4.9 (suspension)

TABLE 3 Preparation of samples containing PBS buffer (pH of 7.3) 25%Stock Solution Sample in PBS (g) PBS Buffer (g) 20% P407/2% otic agent/8.03 1.82 PBS 18% P407/2% otic agent/ 7.1 2.63 PBS 16% P407/2% oticagent/ 6.45 3.44 PBS 18% P407/2% otic agent/ — 2.63 PBS 2% oticagent/PBS — 4.9

One mL samples are individually placed in 3 mL screw cap glass vials(with rubber lining) and closed tightly. The vials are placed in aMarket Forge-sterilmatic autoclave (setting, slow liquids) andsterilized at 250° F. for 25 minutes. After the autoclaving the samplesare left to cool down to room temperature. The vials are placed in therefrigerator and mixed while cold to homogenize the samples.

HPLC analysis is performed using an Agilent 1200 equipped with a LunaC18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrilegradient (1-10 min) of (water-acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30 μLof sample and dissolving with 1.5 mL of a 1:1 acetonitrile watermixture. Purity of the otic agent in the autoclaved samples is recorded.The stability of formulations in TRIS and PBS buffers is compared.

Viscosity measurements are performed using a Brookfield viscometerRVDVII+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition. Only formulations that show no changeafter autoclaving are analyzed.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, are tested using the above procedure to determine theeffect addition of a secondary polymer on the degradation products andviscosity of a formulation containing 2% otic agent and 17% poloxamer407NF after heat sterilization (autoclaving). Stability of formulationscontaining micronized otic agent is compared to non-micronized oticagent formulation counterparts.

Example 10 Pulsed Release Otic Formulations

A combination of doxorubicin and doxorubicin hydrochloride (ratio of1:1) is used to prepare a pulsed release otic agent formulation usingthe procedures described herein 20% of the delivered dose of doxorubicinis solubilized in a 17% poloxamer solution of example 6 with the aid ofbeta-cyclodextrins. The remaining 80% of the otic agent is then added tothe mixture and the final formulation is prepared using any proceduredescribed herein.

Pulsed release formulations comprising cyclophosphamide, doxorubicin ormicronized thalidomide, prepared according to the procedures andexamples described herein, are tested using procedures described hereinto determine pulse release profiles.

Example 11 Preparation of a 17% Poloxamer 407/2% Otic Agent/78 ppm EvansBlue in PBS

A Stock solution of Evans Blue (5.9 mg/mL) in PBS buffer is prepared bydissolving 5.9 mg of Evans Blue (Sigma Chemical Co) with 1 mL of PBSbuffer (from example 6).

A Stock solution containing 25% Poloxamer 407 solution in PBS buffer isused in this study. An appropriate amount of an otic agent is added tothe stock solution to prepare formulations comprising 2% of an oticagent (Table 4).

TABLE 4 Preparation of poloxamer 407 samples containing Evans Blue 25%P407 in Evans Blue Sample ID PBS (g) PBS Buffer (g) Solution (μL) 17%P407/2% otic agent/ 13.6 6 265 EB 20% P407/2% otic agent/ 16.019 3.62265 EB 25% P407/2% otic agent/ 19.63 — 265 EB

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, are prepared according to the procedures described aboveand are sterile filtered through 0.22 μm PVDF syringe filters (Milliporecorporation), and autoclaved.

The above formulations are dosed to guinea pigs in the middle ear byprocedures described herein and the ability of formulations to gel uponcontact and the location of the gel is identified after dosing and at 24hours after dosing.

Example 12 Terminal Sterilization of Poloxamer 407 Formulations with andwithout a Visualization Dye

17% poloxamer407/2% otic agent/in phosphate buffer, pH 7.3: Dissolve 709mg of sodium chloride (Fisher Scientific), 742 mg of sodium phosphatedibasic dehydrate USP (Fisher Scientific), 251.1 mg of sodium phosphatemonobasic monohydrate USP (Fisher Scientific) and an appropriate amountof an otic agent with 158.1 g of sterile filtered DI water. The solutionis cooled down in an ice chilled water bath and then 34.13 g ofpoloxamer 407NF (Spectrum chemicals) is sprinkled into the cold solutionwhile mixing. The mixture is further mixed until the poloxamer iscompletely dissolved.

17% poloxamer407/2% otic agent/59 ppm Evans blue in phosphate buffer:Take two mL of the 17% poloxamer407/2% otic agent/in phosphate buffersolution and add 2 mL of a 5.9 mg/mL Evans blue (Sigma-Aldrich chemicalCo) solution in PBS buffer.

25% poloxamer407/2% otic agent/in phosphate buffer: Dissolve 330.5 mg ofsodium chloride (Fisher Scientific), 334.5 mg of sodium phosphatedibasic dehydrate USP (Fisher Scientific), 125.9 mg of sodium phosphatemonobasic monohydrate USP (Fisher Scientific) and an appropriate amountof an otic agent with 70.5 g of sterile filtered DI water.

The solution is cooled down in an ice chilled water bath and then 25.1 gof poloxamer 407NF (Spectrum chemicals) is sprinkled into the coldsolution while mixing. The mixture is further mixed until the poloxameris completely dissolved.

25% poloxamer407/2% otic agent/59 ppm Evans blue in phosphate buffer:Take two mL of the 25% poloxamer407/2% otic agent/in phosphate buffersolution and add 2 mL of a 5.9 mg/mL Evans blue (Sigma-Aldrich chemicalCo) solution in PBS buffer.

Place 2 mL of formulation into a 2 mL glass vial (Wheaton serum glassvial) and seal with 13 mm butyl str (kimble stoppers) and crimp with a13 mm aluminum seal. The vials are placed in a Market Forge-sterilmaticautoclave (settings, slow liquids) and sterilized at 250° F. for 25minutes. After the autoclaving the samples are left to cool down to roomtemperature and then placed in refrigeration. The vials are placed inthe refrigerator and mixed while cold to homogenize the samples. Samplediscoloration or precipitation after autoclaving is recorded.

HPLC analysis is performed using an Agilent 1200 equipped with a LunaC18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-95 methanol:acetatebuffer pH 4 gradient (1-6 min), then isocratic for 11 minutes, for atotal run of 22 minutes. Samples are diluted by taking 30 μL of sampleand dissolved with 0.97 mL of water. The main peaks are recorded in thetable below. Purity before autoclaving is always greater than 99% usingthis method.

Viscosity measurements are performed using a Brookfield viscometerRVDVII+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to the procedures described herein, aretested using the above procedures to determine stability of theformulations.

Example 13 In Vitro Comparison of Relase Profile

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm), 0.2 mL of a gelformulation described herein is placed into snapwell and left to harden,then 0.5 mL buffer is placed into reservoir and shaken using a Lablineorbit shaker at 70 rpm. Samples are taken every hour (0.1 mL withdrawnand replace with warm buffer). Samples are analyzed for otic agentconcentration by UV at 245 nm against an external calibration standardcurve. Pluronic concentration is analyzed at 624 nm using the cobaltthiocyanate method. Relative rank-order of mean dissolution time (MDT)as a function of % P407 is determined. A linear relationship between theformulations mean dissolution time (MDT) and the P407 concentrationindicates that the otic agent is released due to the erosion of thepolymer gel (poloxamer) and not via diffusion. A non-linear relationshipindicates release of otic agent via a combination of diffusion and/orpolymer gel degradation.

Alternatively, samples are analyzed using the method described by LiXin-Yu paper [Acta Pharmaceutica Sinica 2008, 43(2):208-203] andRank-order of mean dissolution time (MDT) as a function of % P407 isdetermined.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to the procedures described herein, aretested using the above procedure to determine the release profile of theotic agents.

Example 14 In Vitro Comparison of Gelation Temperature

The effect of Poloxamer 188 and an otic agent on the gelationtemperature and viscosity of Poloxamer 407 formulations is evaluatedwith the purpose of manipulating the gelation temperature.

A 25% Poloxamer 407 stock solution in PBS buffer and the PBS solutiondescribed above are used. Poloxamer 188NF from BASF is used. Anappropriate amount of otic agent is added to the solutions described inTable 5 to provide a 2% formulation of the otic agent.

TABLE 5 Preparation of samples containing poloxamer 407/poloxamer 18825% P407 Stock Poloxamer PBS Buffer Sample Solution (g) 188 (mg) (g) 16%P407/10% P188 3.207 501 1.3036 17% P407/10% P188 3.4089 500 1.1056 18%P407/10% P188 3.6156 502 0.9072 19% P407/10% P188 3.8183 500 0.7050 20%P407/10% P188 4.008 501 0.5032 20% P407/5% P188 4.01 256 0.770

Mean dissolution time, viscosity and gel temperature of the aboveformulations are measured using procedures described herein.

An equation is fitted to the data obtained and can be utilized toestimate the gelation temperature of F127/F68 mixtures (for 17-20% F127and 0-10% F68).

T _(gel)=−1.8(% F127)+1.3(% F68)+53

An equation is fitted to the data obtained and can be utilized toestimate the Mean Dissolution Time (hr) based on the gelationtemperature of F127/F68 mixtures (for 17-25% F127 and 0-10% F68), usingresults obtained in examples above.

MDT=−0.2(T _(gel))+8

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, are prepared by addition of an appropriate amount of oticagents to the solutions described in Table 5. The gel temperature of theformulations is determined using the procedure described above.

Example 15 Determination of Temperature Range for Sterile Filtration

The viscosity at low temperatures is measured to help guide thetemperature range at which the sterile filtration needs to occur toreduce the possibility of clogging.

Viscosity measurements are performed using a Brookfield viscometerRVDVII+P with a CPE-40 spindle rotated at 1, 5 and 10 rpm (shear rate of7.5, 37.5 and 75 s⁻¹), equipped with a water jacketed temperaturecontrol unit (temperature ramped from 10-25° C. at 1.6° C./min).

The Tgel of a 17% Pluronic P407 is determined as a function ofincreasing concentration of otic agent. The increase in Tgel for a 17%pluronic formulation is estimated by:

ΔT_(gel)=0.93[% otic agent]

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to procedures described herein, aretested using the above procedure to determine the temperature range forsterile filtration. The effect of addition of increased amounts of oticagent on the Tgel, and the apparent viscosity of the formulations isrecorded.

Example 16 Determination of Manufacturing Conditions

TABLE 6 Viscosity of potential formulations at manufacturing/filtrationconditions. Apparent Viscosity^(a) (cP) Temperature Sample 5° C. belowTgel 20° C. @ 100 cP Placebo 52 cP @ 17° C. 120 cP   19° C. 17% P407/2%otic 90 cP @ 18° C. 147 cP 18.5° C. agent 17% P407/6% otic 142 cP @ 22°C.  105 cP 19.7° C. agent ^(a)Viscosity measured at a shear rate of 37.5s⁻¹

An 8 liter batch of a 17% P407 placebo is manufactured to evaluate themanufacturing/filtration conditions. The placebo is manufactured byplacing 6.4 liters of DI water in a 3 gallon SS pressure vessel, andleft to cool down in the refrigerator overnight. The following morningthe tank is taken out (water temperature 5° C., RT 18° C.) and 48 g ofsodium chloride, 29.6 g of sodium phosphate dibasic dehydrate and 10 gof sodium phosphate monobasic monohydrate is added and dissolved with anoverhead mixer (IKA RW20 @ 1720 rpm). Half hour later, once the bufferis dissolved (solution temperature 8° C., RT 18° C.), 1.36 kg ofpoloxamer 407 NF (spectrum chemicals) is slowly sprinkled into thebuffer solution in a 15 minute interval (solution temperature 12° C., RT18° C.), then speed is increased to 2430 rpm. After an additional onehour mixing, mixing speed is reduced to 1062 rpm (complete dissolution).

The temperature of the room is maintained below 25° C. to retain thetemperature of the solution at below 19° C. The temperature of thesolution is maintained at below 19° C. up to 3 hours of the initiationof the manufacturing, without the need to chill/cool the container.

Three different Sartoscale (Sartorius Stedim) filters with a surfacearea of 17.3 cm² are evaluated at 20 psi and 14° C. of solution

1) Sartopore 2, 0.2 μm 5445307HS-FF (PES), flow rate of 16 mL/min

2) Sartobran P, 0.2 μm 5235307HS-FF (cellulose ester), flow rate of 12mL/min

3) Sartopore 2 XLI, 0.2 μm 54453071S-FF (PES), flow rate of 15 mL/min

Sartopore 2 filter 5441307H4-SS is used, filtration is carried out atthe solution temperature using a 0.45, 0.2 μm Sartopore 2 150 sterilecapsule (Sartorius Stedim) with a surface area of 0.015 m² at a pressureof 16 psi. Flow rate is measured at approximately 100 mL/min at 16 psi,with no change in flow rate while the temperature is maintained in the6.5-14° C. range. Decreasing pressure and increasing temperature of thesolution causes a decrease in flow rate due to an increase in theviscosity of the solution. Discoloration of the solution is monitoredduring the process.

TABLE 7 Predicted filtration time for a 17% poloxamer 407 placebo at asolution temperature range of 6.5-14° C. using Sartopore 2, 0.2 μmfilters at a pressure of 16 psi of pressure. Estimated flow rate Time tofilter 8 L Filter Size (m²) (mL/min) (estimated) Sartopore 2, size 40.015 100 mL/min 80 min Sartopore 2, size 7 0.05 330 mL/min 24 minSartopore 2, size 8 0.1 670 mL/min 12 min

Viscosity, Tgel and UV/Vis absorption is check before filtrationevaluation. Pluronic UV/V is spectra are obtained by a Evolution 160UV/Vis (Thermo Scientific). A peak in the range of 250-300 nm isattributed to BHT stabilizer present in the raw material (poloxamer).Table 8 lists physicochemical properties of the above solutions beforeand after filtration.

TABLE 8 Physicochemical properties of 17% poloxamer 407 placebo solutionbefore and after filtration Viscosity^(a) @ 19° C. Absorbance @ SampleTgel (° C.) (cP) 274 nm Before filtration 22 100 0.3181 After filtration22 100 0.3081 ^(a)Viscosity measured at a shear rate of 37.5 s⁻¹

The above process is applicable for manufacture of 17%. P407formulations, and includes temperature analysis of the room conditions.Preferably, a maximum temperature of 19° C. reduces cost of cooling thecontainer during manufacturing. In some instances, a jacketed containeris used to further control the temperature of the solution to easemanufacturing concerns.

Example 17 In Vitro Release of Otic Agent from an Autoclaved MicronizedSample

17% poloxamer 407/1.5% otic agent in TRIS buffer: 250.8 mg of sodiumchloride (Fisher Scientific), and 302.4 mg of Tromethamine (SigmaChemical Co.) is dissolved in 39.3 g of sterile filtered DI water, pH isadjusted to 7.4 with 1M HCl. 4.9 g of the above solution is used and anappropriate amount of micronized otic agent is suspended and dispersedwell. 2 mL of the formulation is transferred into a 2 mL glass vial(Wheaton serum glass vial) and sealed with 13 mm butyl styrene (kimblestoppers) and crimped with a 13 mm aluminum seal. The vial is placed ina Market Forge-sterilmatic autoclave (settings, slow liquids) andsterilized at 250° F. for 25 minutes. After the autoclaving the sampleis left to cool down to room temperature. The vial is placed in therefrigerator and mixed while cold to homogenize the sample. Samplediscoloration or precipitation after autoclaving is recorded.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm), 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL PBS buffer isplaced into reservoir and shaken using a Labline orbit shaker at 70 rpm.Samples are taken every hour [0.1 mL withdrawn and replaced with warmPBS buffer containing 2% PEG-40 hydrogenated castor oil (BASF) toenhance otic agent solubility]. Samples are analyzed for otic agentconcentration by UV at 245 nm against an external calibration standardcurve. The release rate is compared to other formulations disclosedherein. MDT time is calculated for each sample.

Solubilization of otic agent in the 17% poloxamer system is evaluated bymeasuring the concentration of the otic agent in the supernatant aftercentrifuging samples at 15,000 rpm for 10 minutes using an eppendorfcentrifuge 5424. Otic agent concentration in the supernatant is measuredby UV at 245 nm against an external calibration standard curve.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to the procedures described herein, aretested using the above procedures to determine release rate of the oticagent from each formulation.

Example 18 Release Rate or MDT and Viscosity of Formulation ContainingSodium Carboxymethyl Cellulose

17% poloxamer 407/2% otic agent/1% CMC (Hercules Blanose 7M): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.0) in PBS buffer is preparedby dissolving 205.6 mg of sodium chloride (Fisher Scientific), 372.1 mgof sodium phosphate dibasic dihydrate (Fisher Scientific), 106.2 mg ofsodium phosphate monobasic monohydrate (Fisher Scientific) in 78.1 g ofsterile filtered DI water. 1 g of Blanose 7M CMC (Hercules, viscosity of533 cP @ 2%) is sprinkled into the buffer solution and heated to easesolution, solution is then cooled down and 17.08 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. Aformulation comprising 17% poloxamer 407NF/1% CMC/2% otic agent in PBSbuffer is made adding/dissolving an appropriate amount of otic agent to9.8 g of the above solution, and mixing until all the otic agent iscompletely dissolved.

17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7M65): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.2) in PBS buffer is preparedby dissolving 257 mg of sodium chloride (Fisher Scientific), 375 mg ofsodium phosphate dibasic dihydrate (Fisher Scientific), 108 mg of sodiumphosphate monobasic monohydrate (Fisher Scientific) in 78.7 g of sterilefiltered DI water. 0.502 g of Blanose 7M65 CMC (Hercules, viscosity of5450cP @ 2%) is sprinkled into the buffer solution and heated to easesolution, solution is then cooled down and 17.06 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. A17% poloxamer 407NF/1% CMC/2% otic agent solution in PBS buffer is madeadding/dissolving an appropriate amount of otic agent to 9.8 g of theabove solution, and mixing until the otic agent is completely dissolved.

17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7H9): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.3) in PBS buffer is preparedby dissolving 256.5 mg of sodium chloride (Fisher Scientific), 374 mg ofsodium phosphate dibasic dihydrate (Fisher Scientific), 107 mg of sodiumphosphate monobasic monohydrate (Fisher Scientific) in 78.6 g of sterilefiltered DI water, then 0.502 g of Blanose 7H₉CMC (Hercules, viscosityof 5600 cP @ 1%) is sprinkled to the buffer solution and heated to easesolution, solution is then cooled down and 17.03 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. A17% poloxamer 407NF/1% CMC/2% otic agent solution in PBS buffer is madeadding/dissolving an appropriate amount of otic agent to 9.8 of theabove solution, and mixing until the otic agent is completely dissolved.

Viscosity measurements are performed using a Brookfield viscometerRVDVII+P with a CPE-40 spindle rotated at 0.08 rpm (shear rate of 0.6s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 10-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm). 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL PBS buffer isplaced into reservoir and shaken using a Labline orbit shaker at 70 rpm.Samples are taken every hour, 0.1 mL withdrawn and replaced with warmPBS buffer. Samples are analyzed for otic agent concentration by UV at245 nm against an external calibration standard curve. The release rateis compared to the formulations disclosed in above examples, and MDTtime is calculated for each of the above formulations.

Formulations comprising cyclophosphamide, doxorubicin or micronizedthalidomide, prepared according to procedures described above, aretested using the above procedures to determine relationship betweenrelease rate and/or mean dissolution time and viscosity of formulationcontaining sodium carboxymethyl cellulose. Any correlation between themean dissolution time (MDT) and the apparent viscosity (measured at 2°C. below the gelation temperature) is recorded.

Example 19 Application of an Enhanced Viscosity Cytotoxic AgentFormulation onto the Round Window Membrane

A formulation according to Example 2 is prepared and loaded into 5 mlsiliconized glass syringes attached to a 15-gauge luer lock disposableneedle. Lidocaine is topically applied to the tympanic membrane, and asmall incision made to allow visualization into the middle ear cavity.The needle tip is guided into place over the round window membrane, andthe cytotoxic agent formulation applied directly onto the round-windowmembrane.

Example 20 In Vivo Testing of Intratympanic Injection of Cytotoxic AgentFormulation in a Guinea Pig

A cohort of 21 guinea pigs (Charles River, females weighing 200-300 g)is intratympanically injected with 50 μL of different P407-DSPformulations described herein, containing 0 to 50% otic agent. The gelelimination time course for each formulation is determined. A faster gelelimination time course of a formulation indicates lower meandissolution time (MDT). Thus the injection volume and the concentrationof a cytotoxic agent in a formulation are tested to determine optimalparameters for preclinical and clinical studies.

Example 21 In Vivo Extended Release Kinetics

A cohort of 21 guinea pigs (Charles River, females weighing 200-300 g)is intratympanically injected with 50 μL 17% Pluronic F-127 formulationbuffered at 280 mOsm/kg and containing 1.5% to 35% cytotoxic agent byweight of the formulation. Animals are dosed on day 1. The releaseprofile for the formulations is determined based on analysis of theperilymph.

Example 22 Evaluation of Cytotoxic Agent Formulations in an AIED AnimalModel

Methods and Materials

Induction of Immune Response

Female albino National Institutes of Health-Swiss mice (HarlanSprague-Dawley, Inc., Indianapolis, Inc.) weighing 20 to 24 g are used.Keyhole limpet hemocyanin (KLH; Pacific Biomarine Supply Co., Venice,Calif.) is suspended in phosphate-buffered saline (PBS) IpH 6.4),dialyzed aseptically against PBS and centrifuged twice. The precipitate(associated KLH) is dissolved in PBS and injected subcutaneously in theback of the animal (0.2 mg emulsified in Freund's complete adjuvant).The animals are given a booster (0.2 mg KLH in Freund's incompleteadjuvant, and then injected ten weeks later with 0.1 mg KLH in 5 μl PBS(pH 6.4) through a microhole drilled through the cochlear capsule. Thecochlea is approached using an operating microscope and steriletechnique. A postauricular incision is made, and a hole is drilled intothe bullae to allow good visualization of the promontory of the cochlearbasal turn, stapedial artery, and round window niche. The stapedialartery is cauterized and removed, and a 25 μm hole is drilled throughthe cochlear capsule into the scala tympani of the lateral basal turn.KLH or PBS control is slowly injected using a Hamilton syringe coupledwith a plastic tube to a glass micropipette filled with the antigen orcontrol. The hole is sealed with bone wax after injection, and excessfluid is removed. Only one cochlea per animal is treated with KLH.

Treatment

KLH and control mice are sorted into two groups (n=10 in each group).Cytoxic agent formulation containing etanercept, prepared according tomethod of Example 4, is applied to the round window membrane of onegroup of animals. Control formulation containing no methotrexate isapplied to the second group. The cytotoxic agent and controlformulations are reapplied three days after the initial application. Theanimals are sacrificed after the seventh day of treatment.

Analysis of Results

Electrophysiologic Testing

The hearing threshold for the auditory brainstem response threshold(ABR) to click stimuli for each ear of each animal is initially measuredand 1 week after the experimental procedure. The animals are placed in asingle-walled acoustic booth (Industrial Acoustics Co, Bronx, N.Y., USA)on a heating pad. Subdermal electrodes (Astro-Med, Inc. Grass InstrumentDivision, West Warwick, R.I., USA) were inserted at the vertex (activeelectrode), the mastoid (reference), and the hind leg (ground). Clickstimuli (0.1 millisecond) are computer generated and delivered to aBeyer DT 48, 200 Ohm speaker fitted with an ear speculum for placementin the external auditory meatus. The recorded ABR is amplified anddigitized by a battery-operated preamplifier and input to a Tucker-DavisTechnologies ABR recording system that provides computer control of thestimulus, recording, and averaging functions (Tucker Davis Technology,Gainesville, Fla., USA). Successively decreasing amplitude stimuli arepresented in 5-dB steps to the animal, and the recorded stimulus-lockedactivity is averaged (n=512) and displayed. Threshold is defined as thestimulus level between the record with no visibly detectable responseand a clearly identifiable response.

Histochemical Analysis

Animals are anesthsized and sacrificed via intracardiac perfusion ofheparinized warm saline followed by approximately 40 mlperiodate-lysine-paraformaldehyde (4% paraformaldehyde finalconcentration) fixative. Right-side temproal bones are immediatelyremoved and decalcified with buffered 5% ethylenediamine tetra-acetate(pH 7.2) for 14 days (4° C.). After decalcification, temporal bones areimmersed sequentially in increasing concentrations (50%, 75%, 100%) ofoptimal cutting temperature (OCT) compound (Tissue-Tek, Miles Inc.,Elkhart, Ind.), snap-frozen (−70° C.), and cryostat-sectioned (4 μm)parallel to the modiolus. Sections are collected for hematoxylin andeosin (H&E) staining and immunohistochemical analysis.

The severity of inflammation is assessed according to the amount ofcellular infiltration of the scala tympani, and an unbiased score isgiven to each cochlea. A score of 0 indicates no inflammation, and ascore of 5 indicates that all cochlear turns had severe infiltration ofinflammatory cells.

Example 23 Evaluation of Cytotoxic Agent Formulations in an Otitis MediaAnimal Model

Induction of Otitis Media

Healthy adult chinchillas weight 400 to 600 g with normal middle ears,ascertained by otoscopy and tympanometry are used for these studies.Eustachian tube obstruction is performed 24 hours before inoculation toprevent the inoculum from flowing out of the eustachian tube. Onemilliliter of type 3 S. pneumoniae strain at 4-h-log phase (containingapproximately 40 colony forming units (CFU)) is placed directly intoboth middle ear hypotympanic bullae of the chinchillas. Control mice areinoculated with one milliliter sterile PBS.

Treatment

S. pneumoniae inoculated and control mice are sorted into two groups(n=10 in each group). Cytotoxic agent formulation of Example 4containing methotrexate is applied to the walls of the tympanic cavityof one group of animals. Control formulation containing no methotrexateis applied to the second group. The methotrexate and controlformulations are reapplied three days after the initial application. Theanimals are sacrificed after the seventh day of treatment.

Analysis of Results

Auris media ear fluid (MEF) is sampled at 1, 2, 6, 12, 24, 48 and 72hours after pneumoccal inocualtion. Quantitative MEF cultures areperformed on sheep blood agar, with the quantitation threshold set at 50CFU/ml. Inflammatory cells are quantitated with a hemocytometer, anddifferential cell enumeration performed with Wright's staining.

Example 24 Evaluation of Cytotoxic Agent Formulations in an Ear CancerAnimal Model

Cytotoxic agent formulations are tested in an ear cancer animal model,described in Arbeit, J. M., et al. Cancer Res. (1999), 59: 3610-3620. Acohort of K14-HPV16 transgenic mice is divided into control/untreatedand test/treated mice groups for comparison of the effect of cytotoxicagent formulation administration on the development of ear cancer. Thecytotoxic agent formulation of Example 2 is administered to the ear ofthe test mice group starting at age 4 weeks. The chemopreventive effectof the cytotoxic agent formulation is assessed by sacrificing treatedmice at age 8, 16, and 32 weeks, and comparing the number of lesions andhistopathological and phenotypic markers (papillomatosis, dermalinflammatory cell infiltration, corneal parakeratosis, etc.) at thevarious stages of neoplastic progression to control mice of the sameage. The effect of cytotoxic agent formulations on the progression ofestablished, late-stage neoplasia is assessed by administering thecytotoxic agent formulation of Example 2 to K14-HPV16 transgenic micestarting at age 28 weeks. The mice are sacrificed at age 32 weeks, andthe effect of the cytotoxic agent formulation is assessed by comparingthe number of lesions and histopathological and phenotypic markers tocontrol mice of the same age.

Example 25 AIED Clinical Trials Using Cytotoxic Agent Formulations

Ten adults patients are selected due to initial steroid responsivenessfollowed by recurrence of hearing loss when steroids are tapered orafter completion of steroid treatment. The cytotoxic agent formulationof Example 4 containing methotrexate is administered to each patient'sround window membrane through piercing of the tympanic membrane.Reapplication of the cytotoxic agent formulations is performed 7 daysafter the initial application, and again at 2 and 3 weeks of treatment.

Hearing evaluations consisting of pure tone audiometry (250-8,000 Hz)and speech testing using dissyllabic word lists in French areadministered to each patient. Testing is carried out both before theapplication of the cytotoxic agent formulation and at 1, 2, 3 and 4weeks post-initial treatment.

While preferred embodiments of the present invention have been shown anddescribed herein, such embodiments are provided by way of example only.Various alternatives to the embodiments described herein are optionallyemployed in practicing the inventions. It is intended that the followingclaims define the scope of the invention and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

1. A pharmaceutical composition or device comprising an amount of acytotoxic agent that is therapeutically effective for treating an oticdisease or condition, the pharmaceutical composition or devicecomprising substantially low degradation products of the cytotoxicagent, the pharmaceutical composition or device further comprising twoor more characteristics selected from: (i) between about 0.1% to about10% by weight of the cytotoxic agent, or pharmaceutically acceptableprodrug or salt thereof, (ii) between about 14% to about 21% by weightof a polyoxyethylene-polyoxypropylene triblock copolymer of generalformula E106 P70 E106; (iii) sterile water, q.s., buffered to provide apH between about 5.5 and about 8.0; (iv) multiparticulate cytotoxicagent; (v) a gelation temperature between about 19° C. to about 42° C.;(vi) less than about 50 colony forming units (cfu) of microbiologicalagents per gram of formulation, and (vii) less than about 5 endotoxinunits (EU) per kg of body weight of a subject.
 2. The pharmaceuticalcomposition or device of claim 1, wherein the composition comprises: (i)between about 0.1% to about 10% by weight of the cytotoxic agent, orpharmaceutically acceptable prodrug or salt thereof, (ii) between about14% to about 21% by weight of a polyoxyethylene-polyoxypropylenetriblock copolymer of general formula E106 P70 E106; (iii)multiparticulate cytotoxic agent; and (iv) a gelation temperaturebetween about 19° C. to about 42° C.
 3. The pharmaceutical compositionor device of claim 1 wherein the composition or device provides apractical osmolarity between about 200 and 400 mOsm/L.
 4. Thepharmaceutical composition or device of claim 1 wherein the compositionor device provides a practical osmolarity between about 250 and 320mOsm/L.
 5. The pharmaceutical composition or device of claim 1, whereinthe cytotoxic agent is released from the composition or device for aperiod of at least 3 days.
 6. The pharmaceutical composition or deviceof claim 1, wherein the cytotoxic agent is released from the compositionor device for a period of at least 5 days.
 7. The pharmaceuticalcomposition or device of claim 1, wherein the pharmaceutical compositionor device is an auris-acceptable thermoreversible gel.
 8. Thepharmaceutical composition or device of claim 1, wherein thepolyoxyethylene-polyoxypropylene triblock copolymer is bioeliminated. 9.The pharmaceutical composition or device of claim 1, further comprisinga penetration enhancer.
 10. The pharmaceutical composition or device ofclaim 1, further comprising a dye.
 11. The pharmaceutical composition ordevice of claim 1, wherein the cytotoxic agent is in the form of aneutral molecule, free acid, free base, a salt, a prodrug, or acombination thereof.
 12. The pharmaceutical composition or device ofclaim 11, wherein the cytotoxic agent inhibits vasopressin receptorfunction, prostaglandin receptor function or estrogen-related receptorbeta function, or combinations thereof.
 13. The composition or device ofclaim 1, further comprising the cytotoxic agent, or pharmaceuticallyacceptable salt thereof, prodrug or combination thereof as an immediaterelease agent.
 14. The pharmaceutical composition or device of claim 1,wherein the cytotoxic agent comprises multiparticulates.
 15. Thepharmaceutical composition or device of claim 1, wherein the cytotoxicagent is essentially in the form of micronized particles.
 16. Thepharmaceutical composition or device of claim 1, wherein the cytotoxicagent is in the form of micronized cytotoxic agent powder.
 17. Thepharmaceutical composition or device of claim 1, wherein the pH of thecomposition or device is between about 6.0 to about 7.6.
 18. Thepharmaceutical composition or device of claim 1, wherein the oticdisease or condition is Meniere's disease, sudden sensorineural hearingloss, noise induced hearing loss, auto immune ear disease or tinnitus.19. A method of treating an otic disease or condition comprisingadministering to an individual in need thereof an intratympaniccomposition or device comprising a therapeutically effective amount ofthe cytotoxic agent, the intratympanic composition or device comprisingsubstantially low degradation products of the cytotoxic agent, theintratympanic composition or device further comprising two or morecharacteristics selected from: (i) between about 0.1% to about 10% byweight of the cytotoxic agent, or pharmaceutically acceptable prodrug orsalt thereof, (ii) between about 14% to about 21% by weight of apolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106; (iii) sterile water, q.s., buffered to provide a pHbetween about 5.5 and about 8.0; (iv) multiparticulate the cytotoxicagent; (v) a gelation temperature between about 19° C. to about 42° C.;(vi) less than about 50 colony forming units (cfu) of microbiologicalagents per gram of formulation, and (vii) less than about 5 endotoxinunits (EU) per kg of body weight of a subject.
 20. The method of claim19, wherein the cytotoxic agent is released from the composition ordevice for a period of at least 3 days.
 21. The method of claim 19,wherein the cytotoxic agent is released from the composition or devicefor a period of at least 5 days.
 22. The method of claim 19, wherein thecytotoxic agent is essentially in the form of micronized particles.